Staining Brains (immunofluorescence) - Any tips to improve staining? (Oct/18/2005 )

Hi all,

I am involved in a study of finding where a toxin binds on the CNS, and I use a tagged recombinant toxin for the study, so I can use immunofluorescence on it. The counterstain is DAPI (premixed into the mounting media - from Molecular Probes).

The staining I am getting is no good, and does not stain too well, if at all. I generally know where it stains now, but I just want to get better pics for publications. Also, the structures are quite badly preserved after cryosectioning. Anyone with tips to improve the general morphology? Please? with cherries on top?

Loads of thanks!!

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So there is alot of info you're leaving out. Like what the tag is and what fluorochrome you're using and if your tissue is fixed or not. Briefly, if you run down your methods for both cryosectioning and staining, I might be able to give some suggestions.

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Hi Cynkern,

Sorry for the lack of information. It's as follows:

It's a oligohistidine tag that I am using, the protein is expressed recombinantly with a pET32a vector in BL21. Primary Ab is a goat anti-HIS, and followed by an anti-goat secondary conjugated with an Alexa488 (FITC). I am not fixing the tissue (brain), but after extraction it is just washed with dissection buffer (Tris acetate, pH7.4 50mM EDTA, with protease inhibitors), then snap frozen in liquid nitrogen.

The frozen tissue is then mounted with OCT on a cryochuck. After the whole tissue is coated with OCT and whitened over, the whole chuck (just the chuck, not the OCT/ tissue) was immersed in liquid nirogen, then rested in the cryostat for 5 - 10 minutes to equalise the temperature (-20 to -25 degrees Celcius), and sliced to 15 micron thickness. The angle of cut was set to between 0 to 5 degrees, blade set firmly in the cutter, antiroll plate set as much as possible to an optimal setting (although I am not really sure whether there is a good way to confirm it). This is done with a Leica CM1850.

In the in vivo studies, after the toxin was injected and brain processed, the slices are subjected to 2x PBS wash, then the primary incubation (1:1000 in PBS) overnight in the cold room, then washed 3x (15 minutes each) in PBS, then secondary incubation (1:500) in the dark for 4 - 6 hours 4 degrees. Washing 3x 15min each in PBS, then left in the dark chamber to semi-dryness, before the mounting media (Gold Antifade from MP) was put on the slide and mounted with a long coverslip and placed in the cold room until I proceed with confocal.

Hope this is useful. Thanks for the quick reply!!

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You are going to want to fix with something after sectioning but before staining. You also may want to get thinner sections than 15 um. Also do not let dry to semi-dryness before coverslipping.

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Thanks MaximinaNYC,

The thin sections have been giving me the trouble, since they are unfixed. But I am doing about 8 to 10 microns now.

Anyway, I cannot fix them as I am binding the potential receptors with a protein before that. So if I fix them, then I get false positives with the unbound proteins. Is there any way to prevent that?

Thanks.

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