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Author Topic: lymphocyte antigens on paraffn-embedded mouse tissues  (Read 4864 times)

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Offline ZR

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lymphocyte antigens on paraffn-embedded mouse tissues
« on: May 04, 2008, 09:25:09 AM »
Could you recommend any primary antibodies and matching detection kit(s) suitable for basic characterisation of _murine_ lymphomas in _paraffin embedded_ tissues?

I am looking for mouse equivalents of human basic pan-B and pan-T antigens, like CD20, CD79a, PAX5, CD3, CD5, CD2, CD7, etc.

As a 'human' pathologist professionally interested in lymphomas and only a hobby researcher, I found it surprisingly difficult to find, at least among the suppliers normally used for 'human' immunohistochemistry I have contacts with.  Most anti-mouse antibodies listed in catalogues are usually not suitable for archival sections, and there is a 'mouse-on-mouse problem' with detection of tissue-based murine immunoglobulins by secondary antibodies in kits....

lymphocyte antigens on paraffn-embedded mouse tissues
« on: May 04, 2008, 09:25:09 AM »

Offline excalibur

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #1 on: May 04, 2008, 09:58:39 AM »
I use many antibodies raised in rabbit to stain human and mouse tissue.

There are many antibody search engines such as Biocompare, as well as this one on IHC World: http://www.antibodybeyond.com/abfinder/absearchengine.htm

Why would archival tissue be a problem?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline CanuckPhD

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #2 on: May 05, 2008, 01:45:33 AM »
Surprisingly, there are not many antibodies that work on mouse FFPE tissue with respect to lymphocyte markers. There are many for frozen tissue that work great but when you switch to paraffin you only have a handful. I have used rat anti- CD20 and CD5 (both Serotec) that are okay and a rabbit anti-CD3 from Cell Marque.

Good luck

Offline ASluo

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #3 on: May 05, 2008, 08:10:52 PM »
Hi there,

CD3, F4/80 (macrophage) as well as FoxP3 (regulatory T cells) antibodies will work on FFPE mouse tissues. However, antigen retrieval methods and antibody concentration will affect their capacity to stain.

Offline ZR

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #4 on: May 06, 2008, 09:54:01 AM »
Surprisingly, there are not many antibodies that work on mouse FFPE tissue with respect to lymphocyte markers. There are many for frozen tissue that work great but when you switch to paraffin you only have a handful. I have used rat anti- CD20 and CD5 (both Serotec) that are okay and a rabbit anti-CD3 from Cell Marque.

Many thanks for that hint -- I will check the details.

May I know what detection system do you use?  'Human' kits are usually based on a secondary antibody specific for mouse (or broadly speaking, rodent) immunoglobulins.  In such a scenario the secondary antibody binds as a primary to mouse tissue and obscures true stainig of cytoplasmic and membrane antigens of B cells.  Of course this itself can be used as a crude B cell detection system, but there is still an issue of T cells...

Offline excalibur

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #5 on: May 06, 2008, 11:50:01 AM »
Universal kits for human tissue will be a cocktail of mouse and rabbit secondaries. You cannot use these on mouse tissue. You will need to use a kit for your specific antibody host. Most of the antibodies you will find will be raised in rabbit for your mouse tissue. I recommend Vector's ImmPress Rabbit kit. It uses the new polymer technology, so avidin/biotin background staining is not a factor.

I do not have any affiliation with Vector, other than being a happy customer.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline CanuckPhD

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #6 on: May 07, 2008, 01:42:01 AM »
I have had good luck just using a specific anti-rat and anti-rabbit form Jackson (highly absorbed for mouse IgG) biotin conjugated secondary antibody. I have low amounts of background with these antibodies as long as I block in 10% goat serum and quench with H202/TBS/Na azide.

Of course you have to adapt your protocol to your tissue. For example in the kidney and gut there is always more background due to endogenous biotin, whilst in the spleen, lymph nodes and brain (my tissues of interest) there is little endogenous biotin.

Good luck.

Offline ImmunoNYC

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Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #7 on: July 18, 2008, 10:00:29 PM »
B220 is a pan-B marker in mouse and works beautifully on FFPE sections. Use HIER (I use DAKO's TRS and a veggie steamer) and anti-rat secondaries from Jackson ImmunoResearch.
« Last Edit: July 18, 2008, 10:03:22 PM by ImmunoNYC »

Re: lymphocyte antigens on paraffn-embedded mouse tissues
« Reply #7 on: July 18, 2008, 10:00:29 PM »