If you fix with PFA there is no need to fix again in acetone. Some antibodies will not work on PFA fixed tissue / cells and people use acetone to fix post sectioning when they snap-freeze fresh samples. Also, antigen retrieval works best on paraffin sections rather than cryo (you can use more stringent conditions). Have you tried paraffin sections?

We have been using CD11b antibody from BD sciences (BD553309). This will work only in cryo embedded/acetone fixed sections but not the PFA/paraffin embedded tissue sections.

I agree with Dorota; there's no use to fix again after fixing.

You have two ways to process your material:

1. If you want to detect surface antigen in frozen and cryocut samples, just snap freeze in liquid nitrogen and do cryosectioning as usual. The dried sections can be fixed in acetone and after drying they are ready to be stained.

2. If you want to use fixed material you have to get rid of the fixation pior cutting.

By the way ...you can even do cryo cutting after you have fixed it with PFA. Intersting for those who want to examine "the fixed swimming old specimen of the first scientist ever working in this lab" ...

We use 4%PFA as fixing solution for preparing paraffin embedding and doing paraffin cuts. PFA solution is always buffered in PBS at pH 7.2. The rehydration has to be very thoroughly.

Some surface antigens are easily destroyed by fixing with aldehyde containing solutions. Not all of them can be retrieved by antigen retrieval procedure.

Maybe you should switch the antibody for detecting CD68; some clones are not suitable for fixed sections.

Dear Vu Hong, the comments above are correct. Over fixation can also compromise surface markers such as CDs. In general, CD marker stains work better in snap frozen tissue followed by fixation immediately after thaw. While this may work better for surface stain, it typically yields worse morphology... Also, u should pay attention to the specific antibodies u r using, as some may work better with different fixation or tissue preparation methods.... Hope this helps.

I am currently using Iba1 antibody from Abcam on frozen and PFA4% fixed tissue and I don't have any problem. I would not use antigen retrieval in this situation, better for paraffin embedded tissue with extensive fixation. Also, there is no need to fix with cold acetone or methanol since you used PFA. Which antibody are you using? Which protocol?

Fresh frozen sections usually don't have a good morphology, and that's why people usually fix the tissues prior to cryopreservation. I would not recommend to compromise the quality of your material because the antibody does not work well. The best would be to first talk to the company you have purchased your antibody from and see if technical support has any tips for you. If this does not work, look at references where immunos for CD11b and CD68 have been performed and see what antibodies (cat# and company) they have used on mouse tissue and their procedure. Two minor points 1) 5 min post fixation after air-drying prior to staining (either in acetone, methanol or 4% PFA) is standard, and is not considered "overfixation"; better not to skip this or your sections may float in the next incubations. 2) Antigen retrieval is for paraffin sections. If your antibody does not work on fixed-frozen sections it will most likely not work on paraffin sections either, even with antigen retrieval. In frozen sections the epitopes are more exposed than in paraffin but compared to paraffin you lose in morphology. It's a compromise to get better staining.

Make sure you also look at your protocol for washing the sections. When looking for staining of a surface antigen using detergents such as Tween or TX100 in your wash, you may remove any possible staining and you'd never see a signal. As Nicolas Stigani stated above, it would be good practice to try the antibody on positive control tissues first to make sure the antibody works.

Acetone fixation often perturbs membranes, and cell surface markers may be lost. I have had good luck with 4% PFA fixation, sucrose, then cryosectioning onto coated slides. This prevents the tissue from floating off during antigen retrieval. I have also used antigen retrieval (HIER pH 6.0) successfully on these sections. One key point is to make sure you are blocking your sections after antigen retrieval, and you may want to try a blocking buffer without detergents, as was mentioned above. I use the fish skin blocking buffer I've attached. You could easily omit the triton from this, if necessary. Also, a good positive control will help you - can you over-express your protein of interest in a heterologous cell line and test for staining? This will at least tell you if the antibody can detect the protein. Not all antibodies are created equal. Good luck!

There is a fixative called Glyo-fixx that is glyoxal based and provides much better antigen preservation. This is definitely my fixative of choice nowdays and I would highly recommend trying it for antibodies that you have trouble immunostaining following PFA fixation. It works fine for tissue that needs cryoprotecting and cutting.

Thank you very much for all of your valuable suggestions and comments!

I currently use antibody CD68 from Serotec and CD11b from Abcam. To prevent the detachment of sample, I coated the slides with Poly-L-lysin and the samples look ok after antigen retrieval even with autoclaving method. Blocking with BSA 5% in TBST and wash with BSA 1% in TBST, antibodies were also diluted in BSA 1% in TBST.

Both of the antibodies can detect the antigens (CD68 or CD11b) well but in different fixation protocol I said above (I can see they stained the target cells very clearly, brightly and specifically). I am still finding the suitable fixation protocol for both of them. If fail again, I may try with snap-frozen or Glyo-fixx.

Thanks again for all of your kind words ^^