I am analyzing the tissue damage to the mouse retinal epithelial tissue during an eye infection and I need a quality cross section of the eye for histological analysis. I would prefer small sections, I would like to do 12 um, but the sections shrivel up when the blade hits it. I was wondering if anyone had any ideas for troubleshooting this problem? I have removed the lens prior to sectioning. Thanks
I got the best results (in eye lens) as follows: previously frozen the tissue at -40 C (and not at -80C), placed in the cryostat at -20 C for 20 minutes, then i made the slices at a low speed with a continuous motion.
http://www.pathologyinnovations.com/index.html
This page is very very useful for cryosectioning. It shows freezing and cutting technics.
"Shrivel" sounds for me, that the cutting temperature is too low. Thy eyeball is very aequous and I can imagine, that cutting it is like cutting an iceblock. Warming the block surface witha gloved finger helps to get a flatten section.
The fixation is quite important too, I used 20 min in PFA4% in PBS, after I washed 1x with PBS and did 30 min 5% sucrose in PBS and +- 1h in 30% sucrose in PBS, then I freeze it on OCT. Cutting eyes is not easy. The orientation is also important specially if the lens is in the eyes, so place the eye in the way that the lens is perpendicular to the knife. Put the sample for some minutes in the cryostate is important, but is also important to have a nice glass (anti roll plate) on the cryostate, and then the usual stuff the temp (-18 to -21C), the cutting angle (4-6 degrees) and the glass position.
Fixation of the whole globe in 4% PFA at a pH of 7.4 for a minimum of 4 hours followed by 30% sucrose for cryoproection until the globe sinks. The pH of PFA is is important not just for good tissue preservation but to ensure your para formaldehyde has dissolved completely
If you just do the regular histological analysis, I don't recommend you use cryosection. In most cases, the morphology is not so good with cryo-section especially when fixation is not well done. Formalin fixed Paraffin embedded sections can deliver good morphology, and you can get the reference pathology lab to do the whole process pretty professionally, all you need to do is to enucleate and fix the eyeball. 1 hour later post fixation, make a small excision at the limbus, let the fixative in.
If you really have to do cryosection, follow the above suggestions. My suggestion is, again, make a small excision at the limbus to get fixative into the eye. For mouse eye, I think you don't have to remove the lens, you can try to section through the whole eyeball
if you place the direction of lens towards cutting section it will also helpfull to prevent retina detachment.
For me, cutting at low temperatures (< -20°C) helps. The blade must be fresh and sharp, not damaged. I trim / cut away most of the remaining tissue tek before cutting the eye. also trimming the tissue tek to triangle-shape (tip pointing towards the blade) can help. for me, the duration of fixation is not very important, I usually fix the eye only 15min in 4% PFA and then incubate in 30% sucrose before freezing it.
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