Briefly, I have been troubleshooting a Super Golgi Kit (Bioenno https://bioenno.com/products/supergolgi-kit) in order to adopt the protocol for 40 um thick rat coronal brain slices that underwent brain perfusion in saline and then fixed in 4% PFA for 24 hrs. There are papers describing this tissue and fixation being possible, and in theory, it should be.
The protocol is as follows: wash cryopreservant off with PBS, incubate 1% potassium dichromate, 1% mercuric chloride and 1% potassium chromate-based impregnation in a well-plate on a shaker, then some kind of PBS and water-based washing for two days (renew solution every day), then an ammonium hydroxide solution for 20 min, then post-staining wash buffer for 20 min. The protocol is performed at room temp. After impregnation and first washing, the ammonium solution changes the slices from yellow to grey, but then after the washing a lot of the grey fades away.
I tested impregnation times of 1 day, 3 days, 7 days and 10 days, (14d TBD) refreshing the solution once after 1 day (as per the instructions). In either case, the reactivity seems to be positive for dendrites but only be positive for glial cells and blood vessels yet doesn't stain the neuronal cell bodies nor the long apical dendrites.
I can't figure out why. Theoretically, such a thin slice shouldn't require as much time for impregnation... Maybe something to do with the post-ammonium hydroxide washing. Does the timing of the reaction and the wash matter? In each case, for this protocol is says 20 min each.
Any help would be greatly appreciated. Thanks.
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