You need antibodies that work on paraffin sections. In most cases antibodies that work perfectly on frozen sections do not work on paraffin sections due to cross-linking. Look for the:"IHC paraffin" statement on the antibody data sheet.

Epitope retrieval is usually done by heating the slides to 95oC in sodium-citrate buffer for at least 20 minutes. This should be also stated in the data sheet, since there are antibodies that require trypsin and/or similar protease digestion.

Dear Daxing Gao in our laboratory we have recently demonstrated CD4 & CD8 in the paraffin embedded tissue sections of lungs and  lymphnode  of cows suffering from tuberculosis using the following protocol:

Dewaxing & deparafinization in Biogenex Company AR Common Solution- 70degree Celsius for 10 minutes.

Heat induced epitope retrieval in citrate buffer or Biogenex Company AR-3 Solution - 95 degree Celsius for 10 minutes, followed by HIER in Tris EDTA buffer ( pH-9.0)- 95 degree Celsius for 10 minutes.

It has given excellent results ( unpublished data).

Regarding quenching of endogenous peroxidase we do this step before adding the primary antibody.

 Finally as Dr Korcan Ayata has mentioned the primary antibodies that work in paraffin sections should be procured rather than those used for frozen sections.

Thanks Korcan and Kuldip. I will try the frozen samples.

I have had success staining for CD4 and CD8 using a 0.1 M Borate buffer for heat induced antigen retrieval.  I used this paper when I was trying to get the staining to work: http://www.ncbi.nlm.nih.gov/pubmed/15503814

My primary antibodies were from Santa Cruz, though I ended up doing immunofluorescence and not IHC as you will be doing.  A friend used this paper's protocol later and had similar results with his primary antibodies.

Hope this helps.

CD4 and CD8 in mouse usually do not work in FFPE blocks. If you locate a real good Ab I'm sure the IHC world would love to know.  The epitopes between the two species are different, and they react differently to formaldehyde fixation.  I have only located one vendor for FFPE CD4 IHC marker (not santa cruz) and the results were very light, and needed an amplification step even with O/N incubation at higher concentration of Ab.  I have also tried the Zn fixed tissue for CD4 IHC on mouse tissue.  The staining was OK, but the fixation of the samples has to be less than 48hrs,specimen size 2-3mm, followed by  a formalin free processing run.  You do get better morphology compared to frozens, if that is part of the results needed. If you have the extra time and can spend it optimizing your protocol fro Aqueous Zn fix, it is worth it to see the morphology,  otherwise stick to fozen sectioning for IF and keep the paraffin for the H&E staining. 

The publication Robert cited is for human tissue- Cd4 and Cd8 are optimized for FFPE human tissue. 

For CD4:

http://us.ebioscience.com/mouse-cd4-antibody-efluor-570-4sm95.htm

Also, best protocols are located here for IHC:/ICC

http://us.ebioscience.com/resources/best-protocols/immunohistochemistry-and-immunocytochemistry-protocols.htm

Rob Evans is correct, ebioscience just release a CD4 antibody that works in mouse ffpe. http://us.ebioscience.com/mouse-cd4-antibody-purified-4sm95.htm Currently I believe there is no good CD8 antibody reactive in mouse. Although Pierce claims to have two that do work, one is mouse on mouse (MA5-13473) and therefore not ideal. The other one is rat (MA5-17594) and I have not been able to find any staining that proves it works in mouse ffpe out there in the literature. 

Yes Go to E-biosciences website they  came out with CD4 and CD8 that work in FFPE tissues in mouse in 2015. I've tried lots of CD4/CD8 over 20 years but finally  someone has the right epitope for FFPE.  I work these up in mouse spleen  just as they stated on the website. We use a Bond RX.

@Jamie Erickson

Jamie, can you share your protocol or rough protocol for the 4sm95 from Ebio. My student has tried Ag retrieval in citrate, 0.1M Borate and so far no really convincing staining.

We've had lots of people tell us about that Ebio released this "wonderful" Ab but most haven't actually purchased or used it first hand. We were starting to wonder if it's all just hype...

Any insight would be helpful.

Hi Daxing,

I don't know, if you have still searching and also it is something like advertising, but we have developed a pair of guinea pig Anti-CD4 and rat Anti-CD8a antibodies witch work well in FFPE mouse samples,I have attached our protocol for CD4 in mouse spleen (for CD8 we used the same protocol says my colleague)

here is a link of a CD4 and CD8 co-straining in mouse spleen;

http://www.sysy.com/products/cd-4/360004-ihcp.html

and the links to the single antibodies;

CD4

http://www.sysy.com/products/cd-4/facts-360004.php

CD8

http://www.sysy.com/products/cd-8/facts-361003.php

If you are interested, please contact me and I could arrange a free sample is send to you either from one antibody or from both.

Best regards,

Carsten

What is the best commercially available antibody to detect CD3+/CD4+ and CD3+/CD8+ T-cells in FFPE sections? - ResearchGate. Available from: https://www.researchgate.net/post/What_is_the_best_commercially_available_antibody_to_detect_CD3_CD4_and_CD3_CD8_T-cells_in_FFPE_sections#56a8ced164e9b202258b4567 [accessed Jan 27, 2016].

Hi Carsten,

Your antibodies worked beautifully. I'd like to have free samples to test whether your antibodies would work in our hands. Do you happen to have FITC-conjugated ones? Could you send me a quote for them? 

Thank you!

Jiafen

Hi Jiafen,

no problem, please mail me to [email protected] the delivery address and I will mail you back a discount code for big discount (I have to talk with my boss how big, but it will be quite good).

Carsten

Hi  Mahnaz,

you can find all information in our attached protocol, or in short; we have used Epitope Retrieval Solution (Dako, Cat. No. S11699). We have pre-heated the solution to 97°C in a heat steamer (BRAUN MultiGourmet), transfered the section in the solution, wait till everything has 97°C again. Then we incubated the sections for 35 min at 97°C. After that, we let everything cool down for 20 min (at the end the temperature should have around 60°C.

Of course,we have tested this protocol only for "our" antibodies. If you want to test our antibodies, please contact me.

I hope this protocols is helpful.

Carsten

Hi Carsten,

Do you have an Australian distributor for the CD8 antibody?

Thanks.

Kind regards,

Donna.

Hi Donna,

yes, this would be

http://www.biosci.com.au/

Best regards,

Carsten

 Dear Dr. Carsten,

I am trying to stain CD8+ T cells in the mouse brain. I get very good staining for CD3 but no staining for CD8. I have tried several different suppliers of the antibodies but none of those worked. Could you please provide me a sample of your antibody?

Best Regards

Hi Carsten, we have been struggling to standardize CD4/CD8 IHC staining on mouse paraffin sections. We recently purchased the antibodies recommended by you. Thank you for sharing the detailed protocol. Will post an update after trying them out.