Dear All,
I've been trying to stain brain slices with Anti-phospho-DARPP-32 (pThr34) SAB4504378 antibody with very poor results. The best were with antigen retrieval, but I have a problem with assessing the right time of heating it in 95 C degrees (after 10 min slices crumble up, before this time the staining doesn't work). As antigen retrieval buffer I'm using Tris-EDTA pH 9.
Thanks in advance.
Best regards,
Iwona
Reduce your fixation time and switch to sucrose/cryosectioning protocol it eliminates the antigen retrieval step altogether. I usually did not get good results with the paraffin exempt very basic chemical-based staining such as H&E, silver, PAS, etc.
You need to tell us more about the sample preparation, eg. how is it fixed? Are you sure the antibody is (still) working properly, ie. have you tested it under various other circumstances, postive control etc?
Hallo Iwona, first mount your sections on superfrost plus slides. These sort of slides garantee that your sections don't float away during AG. Second make a delution raw of the primary antibody to find out weather an AG is nessary. 3. you can change your detection system. That means from labeled secondary antiboty to biotinylated secondary AB plus Strepavidoine (POX or fluophore), because this System is more sensitive than two-step- methods. As Mrtin has recommaded use tissues for this test which expresses your Protein of interest. It makes it easier.
Fourth if all those changings do not fit try AG. Take a look into the datasheet. Maybe your will find informations about the buffer for AG either pH6 (Citrate Buffer) or pH 9 (EDTA-Buffer). I used a 800w microwafe and run 3 circals of 5min each. Cool the sections with the Buffer you used for the AG to room temperature, rinse with PBS or TBS and continue with your immuno. Good luck!
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