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!
Hi Dr. Olga Burenkova
I have observed these blurry effects during my histology. It occurs due to uneven distribution of DPX mounting medium throughout the section. I would advise you gently press the slide with coverslip with a tissue following addition of DPX mounting medium. This would allow the mounting medium to distribute evenly and remove the excess amount.
Best of luck.
Thanks,
Oh my, Samir, what an excellent idea! Thank you very much for sharing it! How long do you typically press the slide? Is it just for a moment or for a few seconds? I am certainly eager to try this!
Hi Dr. Olga Burenkova
* Turn over the stained slide and coverslip assembly and gently press for 5-10 seconds to evenly distribute and remove any excess DPX using an absorbent towel or tissue.
Thanks,
Unfortunately, neither pressing slides after their mounting, vertical slides drying before mounting, nor using more fresh (and less viscous) DPX helped. Such a mystery.
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).
Hi Olga Burenkova
These bubbles are present in the block? or is it forming during sectioning?
I do get some bubbles formation but during the PBS washing, it clears out from the slides.
It's good at least you found the right reason now.
Best of luck.
Thanks,
Hi Samir! The bubbles form during sectioning when I place the slice on the slide. I tried to bend and position the slices somehow to decrease this effect and sometimes it helps!