For microwave antigen retrieval, I use a glass Coplin jar. Put your slides in, fill with citrate buffer, no lid. Microwave on 100% power until just starts to boil; ~1 min or so. Then reduce power to 30% for 10 min. Keep an eye on it. You don't want a rapid boil. If you need, add more citrate buffer. After boil, take them out and cool on bench for about 30 min. Wash with buffer a few times then go forward as normal. I use a PAP pen to draw a circle around my tissue and then block and stain within that circle. Block 1 hr with PBS + 3% BSA and maybe a little rabbit or goat serum if you have it, then O/N 4o with primary. Next day wash and then secondary Abs for 1 hr RT. Wash and go forward with whatever method of revealing you are using, i.e. fluorescent or DAB, etc. Hope this helps.

For microwave antigen retrieval, I use a glass Coplin jar. Put your slides in, fill with citrate buffer, no lid. Microwave on 100% power until just starts to boil; ~1 min or so. Then reduce power to 30% for 10 min. Keep an eye on it. You don't want a rapid boil. If you need, add more citrate buffer. After boil, take them out and cool on bench for about 30 min. Wash with buffer a few times then go forward as normal. I use a PAP pen to draw a circle around my tissue and then block and stain within that circle. Block 1 hr with PBS + 3% BSA and maybe a little rabbit or goat serum if you have it, then O/N 4o with primary. Next day wash and then secondary Abs for 1 hr RT. Wash and go forward with whatever method of revealing you are using, i.e. fluorescent or DAB, etc. Hope this helps.

Hi Caro,

I do all my immunohistochemistry on slides now, and often use microwave antigen retrieval. I use solution at either pH6 or pH9 (during optimisation I see which works best) for 10 mins, then cool the slides down in tap water. Don't let them dry out. Is your tissue paraffin embedded?

The next step would be blocking for 30 mins (at least), then the primary. You can choose to leave it in the primary overnight at 4oc, or you can incubate for 1 hour at room temperature. If your primary is made up in your blocking solution you don't need to wash in between your blocking and your primary (just tap off the solution), but you do need to wash with tbs following primary incubation. Then follow with secondary (if you primary isn't biotinylated), then wash, ABC, wash, DAB, for example.

Emily, thank you for your answer. I think I see what I did wrong the first time. I set the microwave to full power and left it to boil for 20 minutes, it makes more sense to reduce the power to avoid rapid boiling and evaporation. Yes, I did find out about the PAP pen, but unfortunately we don't have it.

Hi Kam, thank you for your reply. No, my tissue is not paraffin embedded. We post-fix the brains with 4%PFA and then keep them in sucrose until cryosectioning, after which we keep the sections in 1xPBS and 0.1% sodium azide until staining.

After taking it out of the microwave do you remove the buffer and pour tap water in the staining dish and just let it cool, or do you just take the rack out and leave it under the tap?

Yes, this is the staining protocol we follow as well, however, I was wondering how do you place the solutions on your slides.  I found that people use this hydrophobic pen to draw a circle around and then place the solution on the slice itself. However, we don't have this pen, so one of my labmates suggested to use high vacuum grease instead. 

Another option one of  my workmates suggested was to try placing the sections in eppendorf tubes with citrate buffer in the water bath at high temperature, so this way I don't need to place the sections on slides, and I can follow the next steps using well plates. However, for DAB staining we incubate with phenylhydrazine prior to blocking solution. If I do the antigen retrieval, shall I do the phenylhydrazine incubation prior of after the antigen retrieval? 

I guess my answer is not going to be very much welcome because basically after trial and error of about 6 months I simply gave up with this whole antigen retrieval business. I noticed a few slides benefited from antigen retrieval especially with heat not microwave. Microwave proved to be really bad for our tissues (mouse and rats, various tissues). The sections would often come out of the slides or become wavy and other stainings such as DAPI sometimes would not work. The time constraints also proved difficult as we could not finish the whole immunocytochemistry procedure in one day. The only success I had was with heat (at least 90 degree C) in PBS in a coplin jar for 1 hr. This seemed to be protective of the tissue degradation while giving better antigenicity. However, later we found that it was mainly due to problems with the primary antibody that the stainings did not work normally without antigen retrieval. We also shifted to neutral buffered formalin and found that it is as good as if not better than PFA.

Try to find a good primary antibody then all your problems will be solved.

Regards

Hello Nurul,

Thank you for your detailed reply.

I managed to test the Olig-2 / DAB staining, both without and with antigen retrieval (using the water bath technique: sections placed at 42 degrees in pre-heated citrate buffer for 30min), and they both worked. I tested 1:100, 1:300 and 1:500 and all concentrations also worked. In the past one of my colleagues tested 1:500 and it didn't work for him, so that is why I wanted to test the antigen retrieval method.

Best wishes.