Hello everyone! Qingling here! SIP's over! :D
This is gonna be my last entry!



Alright, I was in the bone marrow lab for my last section.
but however, i will not mention about the bone marrow aspiration
as vanessa had done so in her blog post (group 6).
pssst! its my most enjoyable lab experience ever! :D



In the bone marrow lab, not only do we go down to the ward
for bone marrow aspiration, we also do staining of the slides and viewing and
reporting of the results. So in this entry, i'll describe to you guys the different
stainings!



Maygrunwald-Giemsa stain:
it is a romanowsky stain used in morphologic classification
of hematopoietic cells. Giemsa alone can demonstrate malarial parasites in thick film.
Romanowsky stain consists of methylene blue and oxidation product and eosin Y or eosin B.
The combined action of these dyes will produce the Romanowsky effect yielding a purple colour
to nuclei of leukocytes and neutrophil granules, and pink colour to erythrocytes. M/G stained wedged bone marrow smear tells us the cellularity, quantity, quality of the magekaryocytes. Presence of tumour clumps should be noted when viewing slides. It also stains the erythroid and granulocytic cells, lymphoid, reticulum and plasma cells.

Procedure :
1) Add 1:1 ratio of Maygrunwald stain to buffered water onto the slides.
2) Mix well and leave it for 7 minutes.
3) Wash throughly.
4) Add 1ml of Giema stain adn 9ml of buffered water.
5) Mix well and leave it for 12 minutes.
6) Wash and air dry.

picture taken from http://www.bccancer.bc.ca/NR/rdonlyres/5A3C3A86-4549-4422-8258-BE8AE2635461/3748/adenogiantcell03208501w2.jpg






Iron stain :
The HCl caused ferric iron in hemosiderin to release from protein. The free iron react with potassium ferrocyanide and gives an insoluble blue compound called ferric ferrocyanide. The ferritin which is soluble does not give a positive reaction. An increase in iron stores shows more hemosiderin granules that are often in clumps on the slides. Hemosiderin is stained blue-green in colour while nuclei is stained red.



Iron stain procedure :
1) A control is used together with the first iron stain slide of the day.
2) Fix the slides with methanol for 10-20 minutes.
3) Wash it with water
4) Mix 1ml of HCL and 1ml of potassium ferrocyanide into a testtube and mix.
5) Pour the mixture onto the slides and leave it for 15 minutes.
6) Counterstain with neutral red solution for one minute.
7) Wash and air-dry



Picture taken from http://aphlabs.com/yahoo_site_admin/assets/images/DSCN8716.187192835_std.JPG






Periodic Acid Schiff stain :
It stains a variety of carbohydrates including glycogen which is often found in haemopoietic cells. It is used to differentiate the diagnosis of acute leukemia. Lymphoblast in coarse granules or large block on clear background are PAS positive. Myeloblast and monoblast are PAS negative. The product will be red in colour. Neutrophils are magenta and cytoplasm are red in colour. Normal erythroblasts do not stain PAS. However PAS is strongly positive in erythroleukemia.





Procedure :
1) Fix the slides with methanol for 15-20 minutes.
2) Wash and add periodic acid and stain it for 10minutes.
3) Rinse and AIR-DRY!
4) Add schiff reagent and leave it for 20 minutes
5) Rinse with water
6) Counterstain with Harris Haematoxylin for 10minutes.
7) Run in tap water for 5minutes.





Picture taken from http://www.kidneypathology.com/Im%E1genes/GNM/05-7007.PAS.2.w.jpg



Peroxidase Stain:
It establish and confirm the diagnosis of acute myeloid leukaemia since lymphoblasts are negative. It requires >3% of bone marrow blasts to show peroxidase activity for acute leukemia to classify as myeloid. Myeloloperoxidase deficiency produces clinical picture similar to that of chronic granulocytic disease. Active myeloperoxidase in granulocytes implies that opsonin function is intact. Cytoplasm shows golden yellow granules with nuclei purple in colour. Monocytes, mature and immature granulocytes and many myeloblasts are positive for peroxidase stain. Lymphocytes, megakaryocytes, plasma cells and erythroblasts are negative.



Procedure :
1)Fix with formol ethanol for 1 min
2) Stain with Ortho-tolidine reagent for 7min
3) rinse under tap water
4) Counterstain wil diluted giemsa for 30mins or more.



Sudan Black stain :
It is clinically used as a marker in differentiation of acute myeloid leukaemia from acute lymphoblastic leukaemia. It is more sensitive more less specific than myeloperoxidase stain in identifying myeloblast. Neutrophils, its precursors and eosinophils are stained blue black. Monocytes stain less intensely than neutrophils. Lymphocytes do not stain with Sudan black.
Positive staining is dark brown to black in cytoplasm of mature and immature granulocytes. Megakaryocytes, platelets, erythroblasts, plasma cells, lymphoblasts are negative.
Neutrophils and precursors are strongly positive. Eosinophils show positivity with appearance. Basophils are variable. Myeloblasts activity usually parallels peroxidase activity with a few positibe granules. Monocytes tend to show fine granular staining.


Procedure:
1) Fix with formol ethanol for one min
2) Stain with sudan black solution for 30mins
3) Rinse in 70% ethanol followed by tap water.
4) counterstain with diluted giemsa for 30mins or more.






Feel free to ask any questions! :D

RNA isolation

With reference to Jess's post on 28 October, I will add on about how to isolate RNA from cells.

RNA is isolated from the cells using NucleoSpin RNA Isolation kit.

NucleoSpin RNA Isolation (Note: Centrifuge at room temperature)

1. Prepare DNase reaction mixture in a sterile 1.5ml microcentrifuge tube; for each isolation/reaction add 10ml reconstituted rDNase to 90ml Reaction Buffer for rDNase.

2. Add 350ml Buffer RA1 and 3.5ml b-mercapethanol to the cell pellet and vortex vigorously to lyse the cells. Note: Addition of b-mercapethanol should be done in the hood.

3. Place NucleoSpin Filter columns (violet ring) in a collection tube and filter the lysate through NucleoSpinâ Filter columns (violet ring) to reduce viscosity and clear the lysate, at 11 000 x g for 1 minute.

4. Transfer the flow-through to a 1.5ml micocentrifuge tube and add 350ml of 70% ethanol. Mix by vortexing (2 X 5 seconds).

5. Pipet the lysate up and down 2-3 times and load the lysate to the NucleoSpin RNA II columns (light blue ring) and centrifuge at 11 000 x g for 30 seconds.

6. Add 350ml of Membrane Desalting Buffer (MDB) and centrifuge at 11 000 x g for 1 minute to dry the membrane (desalt silica membrane – desalting step make rDNase digest much more effectively). Note: Discard the flow-through.

7. Add 95ml DNase reaction mixture prepared in step 1 directly onto the center of silica membrane of the column and incubate at room temperature for 15 minutes.

8. (1^st Washing) Add 200ml Buffer RA2 to the column and centrifuge at 11 000 x g for 30 seconds (Buffer RA2 inactivates the rDnase). Discard the flow-through.

9. (2^nd Washing) Add 600ml Buffer RA3 to the column and centrifuge at 11 000 x g for 30 seconds. Discard the flow-through.

10. (3^rd Washing) Add 250ml Buffer RA3 to the column and centrifuge at 11 000 x g for 2 minutes to dry the silica membrane completely. Then place the column in a nuclease-free collection tube.

11. Elute the RNA in 30ml RNase-free water and centrifuge at 11 000 x g for 1 minute.

12. Store RNA in -80^oC.

Li Yinliang Alex 0704894E

TG02 Group 8

2 November 2009

Others - Aptamers

I will be briefly touching on aptamers as they are gaining much attention among scientists that are developing biosensors. I guess it will be good for us to be "updated" to more "new" recognition elements.

Aptamers are DNA or RNA that can recognise and bind to specific targets. You can compare them to receptors or even enzymes because they work based on the lock-and-key relationship with their targets.

To those who have taken DDCT, you would find the technique of producing these aptamers, a.k.a Systematic Evolution of Ligands by Exponential Enrichment (SELEX), familiar.

First, there will be a library of nucleic acids to which the desired targets are introduced, allowing nucleic acid-target complexes to form. Those nucleic acids that do not bind or are weakly bound to the targets will be removed. This cycle will be repeated a few times before the sequences are amplified. You may think of this step as a stringent selection of the best. Amplification is performed by PCR for DNA sequences and RT-PCR for RNA sequences.

RT-PCR may be new to some of us and so, if you are keen, more information can be found in some of the posts by Tiong Han (1 - brief intro & protocol, 2 - RT-PCR probes), Jess (3 - pictures) and myself (4 - intro).

Some consider aptamers better than antibodies (another recognition element) because SELEX typically take 8 weeks or less while antibodies can take up to months! In addition, aptamers also have a wider range of targets because they can differentiate the chirality and structure of molecules. It's no wonder they are gaining so much attention!

Yvonee 0703189A

Histopathology – Special Stains

HELLOS!
it's yet, my turn again for posting. sorry for the really late posting. hahas.
anyway. i'm currently at the staining department of histopathology lab.. so yup, i'm just gonna share about a few of the manual special stains, as well as about this machine, Ventana NexES special stain, which our lab uses for certain special stain orders. =)

Manual Special stain 1: PAS/D
Also known as, Periodic Acid Schiff Diastase.
It is a stain commonly used to differentiate glycogen from the other carbohydrates.

Procedure:

1. Dewax and bring sections to water (i.e, place slides under running water)
2. Defroze diastase (which is normally kept in the fridge for storage), after which cover sections with diastase completely for 30 minutes.
3. Wash well in water.
4. Drip excess water off section, and treat section with 1% periodic acid for 5 minutes.
5. Wash well in water.
6. Drip excess water off section, and treat section with Schiff's reagent for 3 minutes.
7. Wash well in running water.
8. Counter stain nuclei with haematoxylin for 1 minute
9. Blue in water for 5 minutes, and examine under microscope to check quality of stain.
10. Dehydrate and mount slide with Depex.

Any presence of glycogen will be stain magenta on the PAS stained slide while for the PAS/D stained slide, there will be absence of it as glycogen has already been digested by diastase, hence no reaction will occur with schiff's reaction. A control should always be used to display positive results (i.e. sections appear pale pink)

If sections appear as magenta while control section displayed positive result, it shows that there are still presence of glycogen on the section, suggesting possiblilty of glycogen storage disease.

Positive PASD stain. Section stained pale pink instead of magenta.

 

Negative PASD stain, whereby glycogen are still present on section and stained magenta as seen.

 

Picture taken from  http://path.upmc.edu/cases/case435/images/fig04.jpg and http://emedicine.medscape.com/article/941632-diagnosis respectively.

 

Manual Special Stain 2: Mucicarmine Stain – Southgate’s mucin

Mucicarmine Stain (MC stain), is a type of stain which stains mucin. Mucin is a secretion that is produced by various epithelial cells and connective tissue. When mucin is produced in excess, it may suggest possibility of inflammation of epithelial cells or certain intestinal carcinoma.

Carmine will act as a cationic dye in the presence of aluminium, which will bind with mucin.

Procedure:

1. Dewax and bring sections to water (i.e. place slides under running water).
2. Treat slides with Weigert Iron Haematoxylin for 10 minutes.
3. Wash and blue in water for 5 minutes.
4. Stain with mucicarmine solution for 20 minutes.
5. Wash well with water
6. Dehydrate, and mount slides with depex.

Mucin will be stained red, while nuclei will be stained blue.

mucicarmine stain

Picture taken from http://www.ventanamed.com/includes/galleryThumbnail.php?id=12

 

Manual Special Stain 3: Victoria Blue Nuclear fast red stain (VB stain)

This is a stain which stains for HBsAg and elastic fibres.

Procedure:

1. Dewax and bring sections to water (i.e. place slides under running water).
2. Prepare acidified Potassium Permanganate solution
1. 6.5 ml of distilled water + 3 ml of 0.5% potassium permanganate + 0.5 ml of 3% sulphuric acid
3. Treat sections with acidified potassium permangante solution for 5 minutes.
4. Wash well in water.
5. Treat sections with 4% sodium metabisulphite for 1 minute.
6. Wash well in water.
7. Dry slides on hotplate before placing them into Victoria Blue solution for minimum 4 hours, but preferably 24hrs.
8. Differentiate in 70% alcohol until background is clear. (Approximately 1minute)
9. wash well in water
10. Treat section with nuclear fast red stain for 3 minutes.
11. Wash well in water
12. Dehydrate, and mount slides with depex.

HBsAg, elastic fibres, lipofuchsin and mast cells will be stained blue, while cytoplasm and nuclei will be stained red.

 

Manual stain 4: Ziehl Neelsen Stain (ZN or TB stain)

This stain is used to demonstrate acid fast bacteria strains belonging to the genus mycobacterium. Lipid capsule of the acid fast organism will taje up carbol-fuchsin and resist decolourization with a dilute acid rinse. This lipid capsule of the mycobacterium will not be stained by methylene blue during bluing. This stain is also commonly known for detection of mycobacterium tuberculosis, which caused tuberculosis in patients, hence is other wise known as Tuberculosis Stain (TB stain)

Procedure:

1. Dewax and bring sections to water (i.e. place slides under running water).
2. Treat section with commercial TB solution (carbol-fuchsin) for 5 minutes.
3. Wash well in water
4. Differentiate with 1% acid alcohol until carbol fuchsin is remove from section (i.e. section is clear from stain)
5. wash well in water.
6. Counterstain with 1% Loeffler’s methylene blue for 10 seconds
7. wash well in water.
8. Differentiate in 95% alcohol until section turns sky blue colour.
9. Dehydrate, and mount slides with depex.

Acid-fast bacteria strains will be stained bright red, while the background will be stained blue.

ZN stain

Picture taken from http://en.wikipedia.org/wiki/File:Mycobacterium_tuberculosis_Ziehl-Neelsen_stain_02.jpg

 

Ventana NexES special stain machine

It is a computerized barcode-driven special stain stainer that automatically applies staining reagents to the microscopic slides and mixed over the entire section. Staining process is pre-programmed in the software and accomplished by the serial application of reagents, all of which are held on the reagent carousel (otherwise known as reagent loading tray).

Liquid coverslip is applied before each application of the reagent to inhibit evaporation. Quantity and amount of reagent to be applied on each slide is also pre-programmed by the software.

Staining reagent kits are supplied in vials with a barcode labelled carrier, which can be fixed onto the reagent carousel of the machine. The computer ‘recognises’ the barcode and carries out the staining procedure accordingly.

Barcode labels are printed from the software, carrying the biopsy number of the specimen/slide, as well as the barcode specific for performing a particular stain.

Before slides are placed into the machine, it must first be labelled with the barcode labels, and dewaxed. After which, slides are placed into the machine and a special wash solution is added to the slides prior to staining. This aids to prevent sections from drying up.

Appropriate staining kits must be loaded to the reagent carousel, with caps uncapped. Before initialising a run, check that waste bottle is still sufficient for usage, and there is sufficient levels of buffered wash solution and liquid coverslip for the run.

When the machine runs the staining program, it will read off the slide and reagent barcode labels, and information for staining will be downloaded from the system to the staining module to proceed run.

When the run has completed, slides are removed from the machine, and the underside of the slides are gently clean with a gauze to remove residue stain solutions. It is then placed into 95% ethanol to remove excess liquid coverslip, afterwhich, proceeded on to dehydration as per normal. Slides will then be mount with depex, and the machine will be cleaned using a special cleaning kit purchased from the company.

Pictures taken with permission from lab.

that’s all for now~

Cheers,

Ang Yu Hui

0702632A

Reception

Hey everyone! It's my turn (Felicia) to update this blog! ;)

What I'm about to update is on the reception aspect of Haematology.
You may think that the reception does not really play an important role in the entire process of Haematology. In that case, THINK AGAIN!

The reception is one the sub-sections of the Haematology lab and it plays an important role in receiving and processing samples for all the sections in the laboratory. It also handles telephone enquiries from customers for test results. 2 clerks are stationed at the counter. Operating hours for the reception are from 8am to 6pm from Monday to Friday and 8am to 1pm on Saturday.

From 8am to 4pm, each request form received by the counter is clocked-in to initial sample's receiving date and time.

For samples that require the FBC, clerks or technologists manning the counter should check ;

1. That the particulars on the request form tally with the information appearing on the sample label.
2. That the volume of sample is correct and
3. that the tube used is correct.

Once everything is in order, a lab number in ascending order is assigned for FBC samples and written down clearing on the bottom of the left of request form and on the samples tube. The staff who labelled the tube would initial beside the number on the request form. For additional tests, markings are made on the cap of the sample tube so that the technologist is reminded of these tests.

1. O for ESR
2. R for reitculocytes (Retics)
3. MP for blood film malaria (MP)
4. T for HbH Thalassaemia Screening Stage 1

The request forms are logged into the computer using the number assigned. After logging in the request into the computer, the forms together with the samples are brought into the routine lab for processing.

So you see, the reception plays a crutial role in the initial stage of haematological processing! If any data is being logged incorrectly, it may lead to many confusions when being dispatched into the different departments for the required tests.


Felicia
0703345I

Hi all! Qingling here! I apologise for the late post yeah!

This entry will be on Urine Dipstix!

The urine samples are first collected from the reception by the Health Care Attendant.
Afterwhich, they will tally the name and IC number on the container against the request form. After it is done, he/she will assign a lab number, open up the bottle cap and place them on a tray. 2 rows of 5 bottles each. The request form will be placed under the tray. The med tech will check the request forms to ensure the test ordered is correct. Patients with renal failure or request forms from the Renal Dept has to be tested using the dipsticks. Remove one strip from the dipstick bottle and replace the cap. Completely immerse all the reagent areas of the strip in the urine and remove immediately. While removing it, run the edge of the entire length of the strip against the rim of the urine container to remove excess urine. Then, compare the results with the corresponding color charts on the bottle label.

The intended use of the urine dipstick test is to provide tests for glucose, bilirubin, ketone, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocytes in urine.

The tips of the dipstick are impregnated with chemicals which react with abnormal substances in the urine to produce coloured end products. In some of the tests, the depth of the color produced is related to the concentration of the abnormal substances in urine.

Results should be reported in this manner :

For pH, the glomerulur filtrate of blood plasma is usually acidified by renal tubules and collecting ducts from a pH of 7.4 to about 6.0 in the final urine. Both normal and abnormal range is from 5 to 9.

For specific gravity, it measures the urine density. Random urine may vary from 1.001 - 1.035. Normal adults' urine with normal diet and fluid intake will have SG of 1.016 - 1.3022. Urine pH above 1.035 is either contaminated or have high glucose level.

For protein, normal protein excretion does not usually exceed 150mg/24hours or 10mg/100mL in any single specimen. Normally, no protein is detectable in urine. Color matching greater than Trace indicates proteinuria.

For glucose, dipsticks employing the glucose oxidation reaction for screening are specific for glucose but can miss other reducing sugars such as galactose and fructose.

For ketones, it should be negative in normal urine specimens. Detectable level of ketone may occur in urine during physiological stress such as fasting, pregnancy and vigorous exercise. In ketoacidosis, starvation or with other abnormalities of carbohydrate or lipid metabolism, ketones may appear in large amount before serum ketone concentration are elevated.

For blood, the significance may vary among patients. Blood is often but not always found in urine of menstruating females. It is highly sensitive to haemoglobin and thus complements the microscopic examination.

For bilirubin, there should be no bilirubin detected in normal individual. Trace amount are sufficiently abnormal to require further investigations. Thus colours that are unlike the negative or positive colour may indicate that bilirubin-derived bile pigments are present in urine sample and may be masking the bilirubin reaction.

For urobilinogen, it will detect the urobilinogen in concentration as low as 3umol/L in urine. A result of 33umol/L represents the transition from normal to abnormal.

For nitrite, it should not be detected in normal individual. Proportion of positive nitrite tests in cases of significant infection depends on how long the urine specimens were retained in the bladder prior collection.

For leukocytes, normal urine usually have negative result. Positive results are clinically significant and may be found in random specimens from female due to contamination of specimen by vaginal discharge.

Here is what i've drawn in my own notebook. This are pictures of how cells look like under microscope! Its not very bright. Sorry!

Thats basically all! Feel free to ask if you have any doubts! :D

MTS assay

CellTiter 96® AQueous One Solution Cell Proliferation assay (MTS assay)
It is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays.

MTS tetrazolium compound i.e. (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is bioreduced by cells intro a coloured formazan product, which is soluble in tissue culture medium – decreasing toxicity to cells seen with an insoluble product (MTT assay). This conversion is accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells
Phenazine methosulfate (PMS), an electron-coupling reagent, is combined with MTS reagent to form a stable solution due to PMS’s enhanced stability.
The quantitiy of formazan product is directly proportional to the number of viable cells in the culture as measured by the absorbance at 490nm.

For the MTS assay, 5 x 103 cells per well were cultured in 96-well plates and treated with cytotoxic compounds (What I am studying) for 48 and 72 hours respectively. After incubation for specified times at 37°C in a humidified incubator, 25µL of MTS reagent was added to each well and further incubated for 2-4 hours. Absorbance was measured at 490 nm on a micro plate reader.

Advantages
• Fast – Eliminates solubilization of formazan crystals before absorbance reading and MTS is more efficiently bioreduced compared to MTT
• Safe – Requires no volatile (easily evaporated at normal temperatures) solvent to solubilize formazan product

Li Yinliang Alex 0704894E
TG02 Group 8
6 September 2009

Histology - Microtomy

HELLOS.

been experiencing some problems with blogger.. can’t make my postings. but anyway, i’m back to histopathalogy routine lab.. after a month at cytology and a month at processing area of histo lab.. so far what have been shared by the others on histology is staining, processing/trimming, embedding.. i shall touch on microtomy then~ =)

Microtomy, otherwise also known as sectioning, is as we all know from HTECH, the sectioning of tissue blocks using a microtome. After tissues are processed and embedded into paraffin blocks, it is then sent to the microtomy section for sectioning. Each tissue section produced can range from 2-25 microns.

Tissue blocks are first shaved/rough-cut, before it is being sectioned for microscopic viewing. Rough-cutting/Shaving is done at 20 microns per rotation/slice, until the entire surface of the tissue has been exposed. With rough-cutting/shaving, dense and hard tissues which may cause nicks and blunts to the microtome blade can be easily identified, as well as sutures and staples. If tissues are dense and hard, it is will then be pre-treated with RDO (decalcifier) and sectioned last (after all the other tissue blocks have been sectioned), as they might still cause nicks and blunts to the blade. Sutures and staples will be removed if identified. Nicks and blunts on the blade will cause score lines and poor sectioning of the tissue block, which may affect the diagnosis when evaluated by the pathologists.

After blocks are being shaved, it is then placed in 10% fabric softener solution for 5 minutes, and washed in water before placing them on cryoplate for chilling. Chilling of blocks is actually to facilitate fast sectioning, and renders tissue blocks hard for thin sections (normally 3-4 microns) to be produced.

The microtomes are those rotary microtomes, whereby the rotary movement is converted into up and down movement for sectioning, and a forward movement to advance the block for continuous sectioning.

Tissue blocks which are chilled, will be clamped onto the microtome, whereby thickness of the sectioning will be set at 3-4 microns, and section into even thickness and long ribbon of sections. These tissue sections will then be transferred onto the illuminated floatation warm water bath (set at 48 degree Celsius +/- 4 degree Celsius). Tissue sections can also be placed into 1% alcohol floatation bath before transferring to the warm water bath, which serves as an alternative when folds on the tissues are difficult to get rid of. Alcohol have a low vapour pressure which will increase surface tension of the sections, thus aid in the spreading of the tissue sections, so that it can be transferred flat, monolayer onto the microscopic glass slides.

Transferring of the tissue sections onto microscopic glass slides is done by ‘fishing’, whereby the desired sections will be ‘fished’ out of the floatation bath using the glass slides, and subsequently labelled with technican’s (who sectioned the block) initial, accession number (of the specimen), batch number, and block number.

Slides will then be placed on the hot plate (with tissue section facing upwards) for 3 minutes to ensure that the sections will not float off during staining. After which, it will be sent for staining (either H&E by the autostainer, or special stains such as PAS, GMS etc.)

Common faults which may be encountered during sectioning:

1. Ribbons fail to form, which can be due to

• paraffin wax too hard
• blade too blunt
• tilt/angle of blade is too great.

2. Sections compresed, wrinkled or jammed, which can be due to

• Blade too blunt
• Tissue block is still warm
• tile of blade and block is too slight

3. Split ribbon or scratches (score lines) in ribbons, which can be due to

• nicks in the blade
• tilt/angle of blade is too great.
• tissue is too hard
• blade edge is blunt (dull/rounded)

4. Sections full of folds, which can be due to

• floatation bath is too cold/warm
• tissue block is still warm

           Automated Microtome which we use in the lab!

               Controller for the automated microtome (close up)

Cryoplate

Illuminated floatation water bath

Manual Microtome(similar to the one we have in sch!)

Pictures Credit to Lab, taken with permission.

Cheers,

Ang Yu Hui Jacelyn
0702632A

Lab Technique - RT-PCR

RT-PCR stands for real-time polymerase chain reaction. Both PCR and RT-PCR are able to amplify target DNA sequences but the value-addedness of RT-PCR comes from the fact that it can also detect the amplified sequence in real-time. For PCR, agarose gels are required for the detection of the amplified sequence and it can only done after the amplification process. This becomes very time consuming. Because of the increased number of manual steps involved in the detection of PCR products, it is likely that the rate of error due to contamination or other human error is higher than RT-PCR.

(Image taken from http://bio-rad.gene-quantification.info/)

This is a real-time PCR system (CFX96™ Real-Time PCR Detection System from Bio-Rad) that my lab uses. It is able to perform several assays at any one time and is relatively user-friendly.

The procedures to run RT-PCR are easy as the vendor would have provided you with sufficient information to run your samples. For example, for the TaqMan probes that I was using, I was required to add in 10µL of TaqMan Universal PCR Master Mix, 1µL of Assay Mix, desired amount of sample and RNase-free water. The parameters for RT-PCR (not mentioned) and formula are all provided by the vendor. As long as the protocol is followed, every thing should be a-ok.

Lastly, RT-PCR can be used for quantification of gene expression, detection of pathogens and genotyping. There are of course, more applications out there than that that I've listed. It is really how creative anyone can get to exploit this useful machine.

Yvonee Chew 0703189A

P/S Where is Jacelyn?

Bone Marrow Morphology

Hey everyone! Felicia here once again. :)

I'm now attached to the Bone Marrow Morphology Lab for 3 weeks. This lab has the most senior medical technologists and is considered one of the most exciting and challenging labs amongst all the others labs in Haematology. When situated in this lab, one will have the golden opportunity to go down to the wards and experience how the bone marrow is being aspirated out from patients! Indeed it may give one a nauseating feeling, but this is what makes the job in this lab so exciting about. ;)

Bone Marrow Preparation, Staining & Reporting

After clinical history, physical examination and the review of peripheral blood film, bone marrow examination is the next most important step used in the diagnosis of haematological disorders. It is the only way used in the classification of leukaemias and myelodysplasias. It allows the assessment of the body iron stores, marrow cellularity, myeloid to erythroid ratio, maturation of cell lines and relative quantitation of eosinophils, lymphocytes and plasma cells. In addition, infiltration by fibrosis and other malignant cells can also be diagnosed. It is also used to assess response to treatment in leukaemias and to assess prognosis in aplastic anaemia and agranulocytosis. The absence of iron stores help to differentiate iron deficiency anaemia from other hypochromic anaemias. Trephine (a fixative for the bone) biopsy may help to diagnose myelofibrosis, aplasitc anaemia, malignant lymphoma or secondary carcinoma.

Equipments & Materials

1. Microscope
2. Multitimer
3. Differential counter
4. Glass slides
5. Spreader
6. May-Grunwald stain
7. Giemsa stain
8. Buffered distilled water pH8.6
9. Mounting medium DPX
10. Adhesive labels
11. Reporting worksheets

Specimen Collection

The marrow aspirate can be obtained from the posterior superior iliac spine due to its accessibility and relative safety which a sample can be obtained.

Preparation of bone marrow smears

Bone marrow smears can be made directly at the beside during aspiration procedure. The sample is then sent to the lab for slide preparation.

Wedge smears
About 0.3ml of bone marrow from the first stringe is put onto a clean glass slide to allow the blood to drain away onto another slide. The marrow fragments tend to adhere to the slide and most of them will be left behind. A smooth edge spreader of not more than 2cm in width is used to make 10-12 wedge smears of size 2x3cm. The marrow fragments are then dragged behind the spreader and they leave a trail of cells behind them. Differential counts are then performed by the senior staff.

Squashed preparation
A few particles are placed on the centre of a clean glass slide in which another slide is gently compressed to spread and disperse the particles as the slides are pulled apart.

Trephine imprint
Several touch or imprint of the bone fragment are prepared by gently touching the core and rolling between 2 slides.

Handling conditions

1. All bone marrow smears are labeled with the patients' initials immediately after preparation. They are kept in separate trays and brought back to the lab with request forms to be filled in by staff.

2. A unique marrow number is assigned to each patient sample and are written in request forms, record book etc.

3. 2 wedge smears, 1 blood film, 1 squashed preparation and an imprint are selected for May Grunwald Giemsa staining. Another wedge smear with visible particles is used for iron staining. Controls with normal/increased iron stores are stained in parallel with the patients' smear.

Bone Marrow Smear - May Giemsa Stain (x1000)

Image from: pathy.med.nagoya-u.ac.jp/atlas/doc/node76.html

4. A set of buff cards and adhesive labels for storing the smears together with a worksheet with patient's details are prepared.

5. Any unstained smears are kept in closed containers for 2 weeks for further tests, if necessary.

I've only spent 2 days in this lab, so this is whatever I've observed and learnt so far! If you guys have any queries, please do not hesitate to ask. ;)


Signing off,
Felicia
0703345I

HELLO PEOPLE! QINGLING'S BACK! :D

This month, I'm posted to the Thalassaemia section of the lab! The one that Felicia was at before me! I've learnt to perform five tests there and surprisingly it was pretty easy! My colleagues in the same lab as me were awesome! They taught me every test patiently despite me making mistakes along the way! They even left the test for me to perform all by myself whenever the test was ordered! So before i begin, im sure all of us know what a sickle cell looks like right? yes. like this!

Picture taken from

http://repairstemcell.files.wordpress.com/2009/03/sicklecell.jpg

Therefore, in this entry, I’m going to share with you the Sickling Test! It provides a rapid screening test for Hb-S status of an individual and serves as a confirmatory test for the Alkaline Cellulose Acetate (the one that Felicia mentioned in one of the previous post) and Acid Gel electrophoresis. One important characteristic

of this test is that the sickling phenomenon occurs only with low oxygen tension. It will detect if the RBC will change into a sickle shape after mixing with the chemical that will

reduce the amount of oxygen carrying it.

A positive and negative control should be used in this test.

The method is as follows.

First, we have to prepare the reagent, which is a 0.2g of Sodium Metabisulphite. It is measured accurately using the weighing balance. After which, dissolve with 10mL of deionised water and mix it up and down using a pipette. 5 drops of the reagent is then added to 1 drop of anti-coagulated blood on the slide and mixed well. Immediately cover it with a cover-glass and leave for it to dry completely as the mixture may be present outside the cover glass. Seal the sides of the cover-glass with petroleum jelly-parrafin

wax mixture. Ensure that it covers the sides of the cover-glass properly as no oxygen should enter. Incubate at 37C for 2 hours. Finally, examine under microscope.

Sickling test should be negative in a normal individual. However in patients with Hb-S disease,

a marked sickling is usually visible.

One important thing to note while performing this test is that patients on medication of drugs such as phenothiazines dapsone will give a false negative result. Also old reagents or if preparation

is improperly sealed, will also result in false negative result.

- - -

Isn't it simple? :D

If you have any doubts, dont hesitate to ask me!

Immuoprecipitation (IP) using Dynabeads

(Taken from http://images.google.com.sg/imgres?imgurl=http://www.invitrogen.com/etc/medialib/en/images/mainbody/data/image.Par.55626.Image.-1.0.1.gif&imgrefurl=http://www.invitrogen.com/site/us/en/home/brands/Dynal/Magnets.html&usg=__YukqNWjNDOfL_tHRNjAEQyUhOaM=&h=163&w=180&sz=7&hl=en&start=4&tbnid=eqmFkGj0Kb2_cM:&tbnh=91&tbnw=101&prev=/images%3Fq%3Ddynamag%2Bspin%26gbv%3D2%26hl%3Den)

Immunoprecipitation (IP) using Dynabeads are more efficient and reliable.
It has several advantages over the Crosslink Immunoprecipitation method.

• Reduce protocol time as short to 30 minutes only instead of a 2 day experiment
  No need for time-consuming procedures e.g. preclearing of cell lysates, 1 minute spin down each time etc.
• Materials will not be lost from spun-down resins or excess surface during pipetting
• Reduce the comsumption of expensive antibodies
• Fast and gentle method that causes minimal physical stress to target proteins
• Able to work with concentrated proteins solutions; therefore dilution is not required
  

Binding of Antibody (Ab)

1. Completely resuspend Dynabeads by pipetting or rotating on a roller (5 min).
2. Transfer 50 µl Dynabeads to a tube, place on magnet and remove supernatant.
3. Remove tube from magnet and resuspend the Dynabeads in 200 µl Ab Binding &
   Washing Buffer containing your Ab of choice. (Typically 1 - 10 µg Ab, the optimal
   amount needed will depend on the individual Ab used).
4. Incubate 10 minutes with rotation at room temperature.
5. Place tube on magnet and remove supernatant.
6. Remove tube from magnet and wash the Dynabeads-Ab complex by resuspending
   in 200 µl Ab Binding & Washing Buffer.

Immunoprecipitation of Antigen (Ag)

1. Place tube on magnet and remove supernatant
2. Add your Ag-containing sample (typically 100 - 1,000 µl) to the Dynabeads-Ab
   complex and gently resuspend by pipetting.
3. Incubate 10 minutes at room temperature with rotation.
   Place tube on magnet, transfer supernatant to a clean tube.
4. Wash the Dynabeads-Ab-Ag complex 3 times, using 200 µl Washing Buffer for
   each wash. Mix gently by pipetting.
5. Resuspend the Dynabeads-Ab-Ag complex in 100 µl Washing Buffer and transfer
   the suspension to a clean tube. Place tube on magnet and remove supernatant.

Elution of Ab/Ag complex (alternatives A: denaturing or B: non-denaturing)

• A. Gently resuspend the Dynabeads-Ab-Ag complex in 20 µl Elution Buffer. Add 10 µl
  NuPAGE® LDS Sample Buffer / NuPAGE® Reducing Agent mix and incubate 10 minutes
  at 70°C. Place tube on magnet and load supernatant/sample onto a gel. (Alternatively,
  the Dynabeads-Ab-Ag complex can be resuspended in the SDS sample buffer of your
  choice and heated as per your standard protocol prior to gel loading.)
• B. Gently resuspend the Dynabeads-Ab-Ag complex in 20 µl Elution Buffer. Incubate
  2 minutes at room-temperature. Place tube on magnet and transfer supernatant/sample
  to a clean tube.

However although the kit is fast and easy to use, however the yield is relatively little and there is alot of "background noise" due to the presence of heavy and light chains from antibodies (co-elution due to the lack of crosslinking in this protocol, therefore there is antibody contamination).
Background noise - Antibody contamination that leads to the production of a heavy and light chain bands appearing on the blot, hence rendering it hard to determine which band is the protein band i.e. immunoprecipitated.

I am currently trying to optimize this protocol by trying out preclearing of lysate (prevent nonspecific binding), incubating with sample and antibody for a longer period of time (to enhance yield) and crosslinking using DSS to prevent co-elution of antibodies; so as to produce better results in future.

Li Yinliang Alex
TG02 0704894E
Group 8
3 August 2009

Lab Technique - Electrochemistry

Here's something really different for all of you out there. My internship focuses mainly on electrochemistry which is the study of chemical reactions involving the transfer of electrons between electrodes and electrolytes.

(Picture taken from http://www.als-japan.com/1031.html)

Electrodes come in different sizes and differ in the inert material in the middle. This picture shows a few glassy carbon electrodes. For a gold electrode, the middle will be gold instead of grey.

Electrodes are electrical conductors that can be used for various purposes such as electrocardiography (ECG) and chemical analysis using electrochemical methods. There are different types of electrodes available in the market but the ones that I have been using are the gold (Au) electrodes.

In order to ensure that the Au electrodes are fit for use, they are polished to remove unwanted chemical residues from previous use. New electrodes of course, do not need to be polished. Polishing is done using alumina powder on a nylon pad. The electrodes are held perpendicular to the nylon pad (with Au in contact with the pad that has alumina powder.) Then for a desired time (eg 10 min each), these electrodes are polished in a figure of 8 or circular motion. The particles in the alumina powder will remove the residues on the gold surface. In addition, nano-strip can be used too. By incubating the electrodes in nano-strip for 15 min, any remaining organic residues are removed.

So how do you know if the electrodes are clean? Electrochemical signals are measured with an electrochemical workstation. A clean Au electrode should give a high electrochemical signal while a Au electrode covered with residues will give a low signal because these residues will slow down the transfer of electrons between the electrode and electrolyte.

(Picture taken from http://www.uni-muenster.de/Physik.PI/DeCola/equipment.html)

This is a picture of an electrochemical workstation. The electrodes are connected to the workstation with the use of wires so that a small amount of voltage can be passed through.

Yvonee Chew 0703189A

Cytology - Processing Gynaecological Specimens

Heys!

it's already end of week 4 of SIP. 16 weeks more to go. 4 mths more to go!

anyway. i'm attached to the histopathology/cytology department. over at the hospital i'm attached to, histopathlogy actually sort of belong to the same department, though both deals with different type of specimens, but is usually inter-related.

we didn't get to do much during our first week, even though for that whole week we were at histo department. from week 2 onwards, we are all allocated at different sections. as for me, i'm now at cytology lab for 1mth, so for this post, i'll be talking about things that are done at our cytology lab. =)


Cytology is actually the study of cells to aid in the diagnosis of diseases. In our lab, we deal with 2 different groups of specimen, Gynaecological (gynae) and Non-gynaecological (NG) specimens. Gynae specimens are actually just cervical pap smears, while NG specimens refers to body fluids (peritoneal, pleural, pericardial, esophageal washings, bronchial washings/aspirates etc), CSF, sputum, urine, and fine needle aspirations (knee, breast, thyroid, bones etc). I'm now covering both gynae section and NG section, but due to safety reasons, we're actually not allowed to perform hands-on for NG specimens, so yeah, all we can do is observe and learn the techniques. =) I'll cover on the gynae section because that's what i've been doing most of the time.

Gynae specimens usually comes in two different type. Liquid-Based preparation or Conventional Smears.

SIP slides on CYTO - gynae

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=))


Ang Yu Hui
0702632A

Hb Electrophoresis

Hello everyone! Felicia here. I'm posted to the Haematology department during this 5 months of SIP. As what Qingling has mentioned, the Haem department is divided into various sections namely Coagulation, Routine, Hb Electrophoresis, Flow Cytometry, Bone marrow etc. During these 3 weeks, I was posted to the Thalessemia lab which handles mainly Hb Electrophoresis. Initially, I was rather doubtful about my abilities as I was quite weak in my lab work. Plus the fact that I was all alone! Separated from my other 2 friends who had gone to other labs. Nevertheless, I entered the lab with an open mind and managed to learn many things! My colleagues are really nice and friendy. Whenever I needed help, they never hesitated to teach me or guide me along.

One of the routine work I had to do was sample processing. These are the few steps I had to follow:

1. Collect samples from the Haem reception counter.
2. Label request form and sample lab no, ensure patients particulars match with sample, initial.
3. Record patient's data (as well as all results) in Hb Electro worksheet.
4. Set up cellulose acetate electrophoresis.
5. Set up HbH inclusion bodies test.
6. Record patient's FBC from LIS
7. If no result in LIS, run FBC using the analyzer and file results.

Other than sample processing, I had to perform this test called Cellulose Acetate Electrophoresis. This test helps to detect common Hb variants present in the blood, if present. Cellulose Acetate is used as it is easily available, provides sharp resolution of Hb bands in a short time, and permits clearing.

Materials needed:

1. Supre-Heme buffer
2. Helena Hemolysate reagent
3. Ponceau S stain
4. 5% glacial acetic acid

Preparation:

1. Prepare Hemolysate

- pipette 25ul of washed packed cells into a 10x75mm test tube

- add 150ul of hemolysate agent

- mix and stand for 5 min

2. Prepare Titan cellulose acetate plate

- label

- slowly lower to rack containing supreme heme buffer (leave it for 5 min)

3. Prepare electrophoresis chamber

- pour 100ml of Supre-Heme buffer into each of the oter section of the zip zone chamber

- wet 2 disposable wicks in the buffer and drape over each support bridge and verify contact with buffer

Procedures:

1. Add 200ul of patients' sample into 10x75mm test tubes
2. Wash the cells by adding 0.9% saline till 3/4 full
3. Centrifuge at 3000rpm for 10 min
4. Repeat steps 3 & 4 to obtain packed cells
5. Dispense 25ul of hemolysate reagent in another set of 10x75mm test tubes
6. Add 25ul of packed cells into each of the test tubes containing hemolysate reagent (Note: Pipette up and down to ensure complete haemolysis)
7. Place 10ul of packed cells containing hemolysate reagent into each sample well of prime applicator
8. Prime applicator by depressing the tips gently into samples wells and blot on filter paper
9. Remove wet titan cellulose acetate plate from buffer and blot once between 2 blotters
10. Prime applicator again and transfer to the wet titan cellose acetate plate
11. Transfer the plate to the electrophoresis chamber with the acetate faced down
12. Electrophoresis the plate for 25 min at 350 volts
13. Staining of Hb bands occurs after 25 mins by placing the plates into Ponceau S stain for 5 min
14. Destain in 3 successive washes of 5% acetic acid (2 min for each wash)

Evalutation of Hb bands:

Hb inspected visually for abnormal Hb bands. Helena Hemo controls act as markers for band identification.

Clinical Significance:

Many Hb variants exhibit similar electrophoretic mobility. Hbs A2, E, C & O cannot be differentiated in this medium. However, HbA2, fraction never exceed 10% of the total whereas HbE fraction of heterozygote amounts to 30-40%. When HbE traits is combined with alpha-thalassemia, the percentage of HbE is lower than the usual 30-40% range. Hb S, D, G, H and I cannot be distinguished in the medium. Therefore, additional confirmatiory tests eg. Citrate Agar Electrophoresis at pH 6.3, solubility test and unstable Hb test are necessary to establish the Hb variant.

Overall, this is one of the common tests I've been doing so far. I also tried many other tests such as Hbh inclusion bodies test etc. But I guess I'll leave that for my other posts! Over the 3 weeks, I've been learning and polishing up my lab skills in Hb Electrophoresis. This has already given me confidence in my lab skills and I'm sure there'll be more chances for me to learn more as I rotate to other labs!

Till the next post! :)

HELLO! This is Qingling! I was posted to the Haematology Lab for this whole period of attachment and there are like 6 sections of the lab which we'll be rotating every month. So for the first month, I was attached to the Coagulation Stat Lab. This is a 24hours routine lab and everyone in there are always very busy. The Stat Lab is quite small for the number of people working in there and there are 3 sections within the Stat Lab! One is the routine tests for coagulation, another is for urinalysis (I don't know why its not in the CCHEM lab but my supervisor says a few years ago, they changed it to allow the Haem Lab to do it) and another one belongs to the Routine Lab where they do the FBC testing. So basically, I was pinned down to the section where we do the routine testings for coagulation such as PT/APTT (commonly done in Stat Lab).

On the first two weeks, I've been using the SYSMEX automated blood coagulation analyzer CA-1500.



Picture from http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1015818~a_langId~e_-111~a_productId~e_182051~a_storeId~e_10001.htm



I feel that this is a great invention! It can do multiple tests such as PT, APTT, D-Dimer Assay, Factor Assays, Protein C Assay, Protein S assay etc. The turnaround time for the tests ordered is fast and it increases productivity rate! However in the Stat Lab, only PT and APTT tests are done. The rest of the tests are sent to the Special Coagulation Lab to do.



So basically when samples arrive, this is what I'll do!

1. Collect samples from the Reception Counter
2. Remove blood tube from biohazard bag
3. Check that the name tally with the request form
4. Ensure correct anticoagulant tube is used (citrated ; light blue)
5. Check that volume is sufficient
6. Assign a lab serial number on the request form and initial name next to it
7. Paste the respective barcode label with correct serial number and date on the blood tube
8. Before spinning, invert tube to look for big clot
9. Centrifuge @ 3000rpm for 3minutes. At the same time, log in request in LIS
10. After centrifuging, place them on the sample rack and put on machine's right rack pool ensuring that the barcode labels are facing the scanner in the machine when running.
11. Press START on the analyzer screen to run the test
12. Record the results on the request form
13. Update results in LIS
    

Therefore the principle of Prothrombin Time (PT) is to screen for abnormalities of factors involved in extrinsic pathway such as factor II, V, VII, X, prothrombin and fibrinogen whereas APTT is a screening test that measures clotting factor of intrinsic pathway such as VIII, IX, XI and XII. These can be easily done by using this automated analyzer to perform the tests! The most commonly ordered tests are PT and APTT. It makes work easier for us because manual PT/APTT is so time-consuming! Remember we did it during the Haem practical? Gosh, it's going to take us hours to complete all the tests ordered! Our lab only do it manually if the first result is out of range and when repeated, result is within the range.

Reference range for PT : 9.0 - 12.5sec
Reference range for APTT : 26.0 - 36.1 sec

The manual test is to confirm the result of the repeated test before recording it in the LIS. However, manual testing is rarely done in our lab!

And thats all for my first few weeks in the Stat Lab! I'll be in the Special Coagulation Lab this week and I'll be learning more other tests!

Till then!

-QINGLING- :D

Immunoprecipitation (IP)

Immunoprecipitation

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Crosslink immunoprecipitation (IP)

Materials and method

Materials

• Protein A/G plus Agarose – protein A/G are used with rabbit/mouse antibodies respectively
• 20X Coupling buffer
• Disuccinimidyl suberate (DSS)
• IP Lysis/Wash buffer
• 100X Conditioning Buffer
• 20X Tris-Buffered Saline
• Elution Buffer
• Non-reducing 5X Lane Marker Sample Buffer
• Spin Columns
• Microcentrifuge Collection Tubes
• Microcentrifuge Sample Tubes
• Control Agarose Resin – used to pre-clear lysates to reduce non-specific protein binding
• Ultrapure water

Note:

• Flow through rate should not exceed 600/300ml when using a 2/1.5ml collection tube respectively; as exceeding these volume may result in back pressure in the spin column/incomplete wash/incomplete elution.
• All resin centrifugation steps should be performed at low speeds i.e. 1000-3000 x g as high-speed centrifugation i.e. > 5000 x g may cause resin to clump and make resuspension difficult.

Method

(i) Binding of antibody to protein A/G plus Agarose

1. Prepare 2ml of 1X coupling buffer for each IP reaction by dilution 20X coupling buffer with ultrapure water
2. Using a cut pipette tip, add 20ml of resin slurry into a spin column and place the column into a microcentrifuge tube and centrifuge at 1000 x g for 1 minute. Then discard the flow through. Note: Gently swirl the bottle of pierce protein A/G plus agarose to obtain an even suspension
3. Wash the resin twice with 200ml of 1X coupling buffer, centrifuge and discard the flow-through.
4. Gently tap the bottom of the column on a paper towel to remove excess liquid and insert the bottom plug.
5. Prepare 10mg of antibody for coupling by adjusting the volume to 100ml with sufficient volume of ultrapure water and 20X coupling bluffer. Note: Add the ultrapure water, 20X coupling buffer and affinity-purified antibody directly to the resin column.
6. Attach the screw cap to the column and incubate on the rotator at room temperature for 30-60minutes; ensuring that the slurry remains suspended during incubation.
7. Remove and retain the bottom plug and remove the cap. Then place the column in a collection tube and centrifuge. Note: Save the flow-through to verify antibody coupling.
8. Wash the resin with 100ml of 1X coupling buffer, centrifuge and discard the flow-through.
9. Wash the resin twice with 300ml of 1X coupling buffer, centrifuge and discard the flow-through.

(ii) Crosslinking of bound antibody
Note: DSS crosslinker is moisture sensitive and incompatible with amine-containing buffers e.g. Tris, glycine. DSS should also be dissolved in DMSO/DMF immediately before use (dilute DSS solution in 1:10 DMSO/DMF to make 2.5mM)

1. Tap the bottom of the column on a paper towel to remove excess liquid and insert the bottom plug.
2. Add 2.5ml of 20X coupling buffer, 9ml of 2.5mM DSS and 38.5ml of ultrapure water to the column to make up 50ml. Then attach the screw cap.
3. Incubate the crosslinking reaction or 30-60 minutes at room temperature on a rotator.
4. Remove and retain the bottom plug and open the bottom plug and screw cap and place the coloumn into a collection tube and centrifuge.
5. Add 50ml of elution buffer to the column and centrifuge. Note: Save the flow-through to verify antibody crosslinking.
6. Wash four times with 200ml of elution buffer to remove non-crosslinked antibody and quench the crosslinking reaction.
7. Wash twice with 200ml of cold IP lysis/wash buffer and centrifuge after each wash. Note: Antibody-crosslinked resin can be stored up to 5 days in IP lysis/wash buffer. For longer storage, store resin in 1X coupling buffer (PBS).

(iii) Antigen immunoprecipitation

Note: If antibody-crosslinked resin was stored in PBS, wash twice with IP lysis/wash buffer and discard the flow-through after each wash.

1. Tap the bottom of the column on a paper towel to remove excess liquid and replace the bottom plug.
2. Add sample to the antibody-crosslinked resin in the column. Then attach the screw cap and incubate the column overnight at 4^oC on the rotator.
3. Remove the bottom plug, loosen the screw cap and place in the column in a collection tube and centrifuge the column. Save the flow through. Note: Do not discard the flow-through until confirming that immunoprecipitatio is successful.
4. Remove the screw cap, and place the column into a new tube. Add 200ml of 1X TBS buffer and centrifuge.
5. Wash sample twice with 200ml IP lysis/wash buffer and centrifuge after each wash.
6. Wash sample once with 100ml of 1X conditioning buffer.

(iv) Antigen elution

1. Add 20ml of elution buffer into the column and incubate for 5minutes at room temperature. Centrifuge.