Hi Juan, In addition to the normal steps for ICH, we do a HCL and a borate buffer incubation step - please see the protocol attached. We use 5um paraffin embedded sections. Good luck.

Hello Juan!

I suppose that you are trying to performance an immunohistochemistry, aren´t you?

I usually do IHC with PBST (phosphate buffer 0,1 M, saline (NaCl 0,9%), and Triton 0,1%), this agent (triton) is neccesary to permeabilize membranes, and I use it for citosolic and nuclear targets. This buffer is used to prepare blocking agent for first step, and to prepare antibody dilutions.

Hi Juan, In addition to the normal steps for ICH, we do a HCL and a borate buffer incubation step - please see the protocol attached. We use 5um paraffin embedded sections. Good luck.

Juan,

The classical methods used an incubation in 1 N HCl at 37C for 30 min, followed by sufficient rinses to bring the pH back to neutral, see papers by RS Nowakowski. The pH drop often denatures other antigens, making double immunostaining potentially problematic (however many antibodies will work).

Another approach is to use antigen retrieval - citrate buffer, pH 6 with heat - either boiling or very close to it, for a few minutes - protocols vary. Again, was out the citrate buffer before blocking. The antigen retrieval method works well for membrane receptors also.

Hope this helps. Best of luck

And that attachment ...

Thank you all girls!! I'll follow your tips, thank you very much! :) if i had any problem then i'd ask you again, thank you :)

I have a protocol... my project is about Alzheimer´s Disease. I can share it to you.

thank you Geovanni! yes, it would be very useful for me! thank you!

We also use a 2N HCl step at 37oC. For mouse brain at 30 microns, we incubate 5-7 minutes. For rat brain, at about 40 microns, about 10 min. For primate tissue at 50 microns, we incubate about 20 minutes. It is really easy to over do the HCl step, producing false positive staining. True Brdu staining is nuclear. If you get "positive" staining that is granular and in the cytoplasm, then you over did the HCl step. Follow HCl with a borate buffer pH 6.

Very useful answer!, thank you! :)