I recently conducted staining on brain sections of adult zebrafish using Nissl stain. The brains underwent pre-fixation in 4% paraformaldehyde, followed by storage in 75% ethanol at -20°C for a period of time, before being rapidly frozen in methylbutane on dry ice in an OCT mold. Cutting was then performed at a thickness of 20 microns at a temperature of -15°C, using charged slides. The stained slides were mounted with DPX and left to dry at room temperature for three days.

Unfortunately, upon examination at 10X magnification, not the entire slice is in focus. I also attempted to use gelatin-treated slides instead of charged ones, which yielded only slightly improved results.

I suspect that these issues may be attributed to two factors: 1) inadequate adhesion of the brain to the slide due to insufficient stickiness of the slide, and 2) the formation of micro-bubbles between the slide and the slice during the cutting process.

Please share your experiences or suggestions regarding this matter. Thank you!

UPD

I finally found a pattern for these unfocused brain areas - they stem from microbubbles formed during sectioning, which I cannot avoid, unfortunately. In the worst situation, when the bubble covers almost the entire area of the slice, the slices are washed off from the slide. In better cases, I observe the unfocused areas (please see the picture).