In paraffin-sections for IHC, I use these key steps

A. Blocking buffer need PBS with serum/s/BSA etc,

B. Primary and secondary antibody dilution needs PBS-T

** PBS-T is made of of D-PBS containing 0.05% Tween-20 .

Yours is exceptional case:

In my opinion, if you replace D-PBS with milli-q water in each step. Since blocking buffer has serum/s, and hence serum will take care of buffering action,

Second, in PBS-T, make in milli-Q with Tween-20 instead of PBS, the detergent is more powerful rather than buffering at this step in fixed tissues. All washings to be done by Milli-Q water-Tween 20 solution and all dilutions of antibodies.

I guarantee, you do one small experiment on 2 slides, it should work according to my guess. Let me know.

Dear Dr. Dufourt,

You raise an interesting question. My guess is that a SSC-based buffer system may work. I haven’t ever used it for IF. Though 2x SSC may be on the high osmolaric side. Why not use 1xSSC? I did do immunostaining in Tris-based buffer (TBS) to omit the presence of phosphate ions. This works fine. SSC has a lower buffering capacity then e.g. TBS.

I hope my answer does help a little.

Loog luck

Duco Kramer, The Netherlands

Dear Juneja,

Are you certain that you can incubate with media that does not only miss buffer, but also salts? Serum and BSA do have a minor buffering capacity, not enough to keep pH at around 7. The lack of ions will most likely result in damaged (ultra) structure and also in (high) background signal.

Please reconsider your answer, try out the double Dutch recipe before you send it over the internet. Don't forget that people with little knowledge of the techniques may be very confused and disinformed by youre statements.

Minor point: You use tween in the incubation buffer, it seems logic to me to have tween in the blocking as well. I guess you do stain for intracellular epitopes. Intracellular structures need to be blocked too, isn't it? Blocking in immunologic techniques not only shields off Fc-binding domains in tissue and cells, they also prevent Ab’s to bind a-specifically to proteins

I do agree, that is why I mentioned to do experiment with 2 slide and find out if it works. To avoid PBS is not normal though. This is exceptional case which wants to avoid PBS. Or try other options before do the bigger experiment. Mine is undocumented option, I have never done it.

Hello Duco, I have done several different IHCs in the paper enclosed. Each antigen retrieval solution was slightly different. But blocking buffer has PBS-T.

Dear Dufourt,

I recommend TBS if you cannot use PBS for immunostaining procedures.

Thanks for your reply, I cant use PBS because my Fish probe do not survive To PBS so i tryied Ab in hybridization buffer and it works but with high background (On at 37°) so now i tryied 2h RT in ssc i Will tell you if it works. Thanks

Hi Jérémy,

With drosophila antenna I'm washing in TNTX buffer (100 mM Tris–HCl, 150 mM NaCl, 0.1 % TritonX-100, pH 7.0). I also have a 2h blocking step in TNTX buffer + 0.5 % Blocking Reagent before adding the antibody. It works really well.

Thanks Gwenn