Help! I recently ran a double stain for cfos and PV using DAB. The tissue went into the process looking great, after the cfos stain- still looked good. However, once the PV stain was completed, the tissue looked fried, for lack of a better word. The tissue is crumpled up and very dry looking, and easily tears. What could be the cause? Any resolutions? Thank you in advance!
did any sections dry out during primary/secondary incubation?
Are you doing free floating or on slide?
Did the dry/cracked look only show up after xylenes or EtoH dehydration?
Those are the first things I think of.
I'm doing free floating and they started looking dry and cracked during primary and secondary incubation- before the xylenes and etoh dehydration.
Hi Sera, free floating double IHC is better looking using fluorescent labeling. http://www.pnas.org/content/suppl/2014/10/31/1413210111.DCSupplemental/pnas.201413210SI.pdf#nameddest=STXT
It may help to post the detailed procedure of your double-stain. Has this procedure worked in prior IHC? Maybe something is wrong with the antibody-buffer?
You certainly don't do dehydration before the second IHC-part?
I agree with Liu completely, it is not necessary to double stain C-fos and PV by using ABC producing brown color at the end and another blue color by AlP. Specially when cytoplasmic staining of PV will also overlap with the nuclear stain of c-Fos. It will be wonderful if you are able to make immunofluorescene
It will be very difficult to do double staining using DAB method on free floating section. It can work fine on fixed sections only. Dry fries and cracks should develop only after alcohol and xylene step, not before. It looks a small problem, just need to follow the procedure from a published paper.
Hi all- thank you for your answers. I have attached a file with the protocol to the question. Any advice is greatly appreciated.
Additionally, we were trying to model the stain found in this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076747/ with some slight variations as we use a rat model.
From the protocol its not clear whether the animals were perfused with PBS followed by 4% paraformaldehyde in PBS at the very first step. This step is very important, after this step brain can be put again into 4% paraformalehyde for 4h or overnight at 4C.
Hi Sara. I have one question, what procedure did you use to slice the brains? Vibratome, microtome, cryostat?
We did use PBS and Para to perfuse the animals and sliced with vibratome
Cutting sections with vibratome means brains must have been embedded in paraffin. And, I believe that paraffin sections should not be cut at a thickness > 7-10 (up to 15-20) micrometer and you can not use them as free floating. The best way will be to cut sections at a thickness of ~10-15 micrometer and mount them on the slides, retrieve the antigen and then proceed with staining, your staining protocol looks fine. Free floating sections are used in case of brains perfused with PBS, fixed with 4% para and post fixed in the same and then cryoprotected in sucrose (~30%) in PBS, otherwise sections will be very fragile and will give all sort of problems.
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