General Research Topics > Neuroscience

Patchy Calbindin 28kDA staining

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MLongley:
Hi everyone,

I'm having a bit of a problem with staining for Calbindin in rat cerebellar free floating sections. The problem I am having is that rather then uniformly stain all Purkinje cells the Calbindin antibodies I have been using seem to stain only patches of Purkinje cells at a time.

It doesn't seem to be a problem of penetration as it this patchiness is very local with neighbouring cells either being strongly stained or not at all. I don't think it is a problem with the antibodies themselves as I have used to antibodies to Calbindin 28kDA, the first a rabbit poly, and the second an mouse mono so I think the chances of both being faulty in the same way seems unlikely.

I have looked at a lot of the literature, Calbindin seems to be uniformly used as a Purkinje cell marker because of its usual reliability, and most of the papers I have read seem to get uniform staining so this makes me think that the problem may lie somewhere in my protocol. I will give a detailed account of my protocol below and if anyone can give me any tips that would be greatly appreciated.

- Under deep anesthesia brains were fixed by transcardial perfusion of 4% PFA in 0.1M PBS.
- Brains were removed and postfixed overnight in perfusate
- Brains were cryoprotected for 24hrs in 20% sucrose in perfusate
- Brains were cut on a sledge microtome at 40microns and collected in PBS
- Sections were incubated for 1hr at RT in blocking solution (0.1M TBS with; 2.5% goat serum (secondarys raised in goat), 0.05% Tween 20, 0.01% Triton X-100 and 1% BSA)
- Incubation in primary antibodies diluted with blocking solution for 14 hours at 4°C
- Wash 3x10 minutes with 0.1M TBS
- Incubate at RT for 2hours in secondary diluted in blocking solution, with some DAPI
- was 3x10 mins in TBS and mount

Thanks in advance for any help anyone can give
Mike

MDeng:
My colleague had this problem too. she did IHC with paraffin section. Is it the problem of fixation?

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