How do I get better tissue sections from the cryostat?

Britanie Blackhurst @Britanie-Blackhurst

08 October 2021 2 3K Report

I am sectioning brain tissue. During this process sometimes the tissue separates from the OCT or there will be bubbles in brain tissue folds that make the section wrinkle more. These can hinder the integrity of the tissue I want to analyze with immunohistochemistry. When preparing tissues to section, what are the most important details in your protocol that can help fix these issues?

Here is the protocol that I use:

Post dissection Preparation

Transfer brain to a 15mL conical tube containing ~10mL 4% PFA.

Refrigerate brains overnight at 4C in PFA.

The following day, decant PFA and replace with ~10mL 30% sucrose.

Refrigerate brains overnight at 4C in sucrose.

The following day, decant sucrose and replace with ~10mL 30% sucrose.

Refrigerate brains in 30% sucrose until ready to embed (at least 24hrs, up to 1-2 weeks).

Embedding Procedure

Decant sucrose and place brain on a kimwipe, gently pat dry to remove residual sucrose.

Fill tissue block about ½ way with OCT.

Using large forceps submerge brain into OCT, fill the chamber the rest of the way with OCT to cover the brain entirely.

Using an Erlenmeyer flask on ice (with aluminum foil covering the opening), puncture the aluminum foil with the Cytocool nozzle and spray Cytocool into the bottom of the flask. (This will prevent any Cytocool from aerosolizing and spraying back at you.)

Pour liquid Cytocool from flask into a large petri dish on ice, fill about ½ way.

Place tissue block with OCT into the large petri-dish with cytocool on ice. Can add more cytocool if necessary until it reaches about ½ way up the tissue block. Allow OCT to set, will turn completely white when frozen.

Wrap frozen tissue blocks with aluminum foil and label with cryo-labels.

Place tissue blocks in a box and store at –80C until ready to section.

Aamir Sharif

After perfusion with 4 % paraformaldehyde or 10% formalin solution. I keep the brain in same solution for one day and then shift it to 15%, 25% and 30% of sucrose. For crysection i take

brain from sucrose solution and cut the brain region rostral/caudal to region of interest (ROI) with a blade.
I put OCT compound on stage and place brain on it. and then apply OCT on all side of brain and then spray liquid nitrogen. Cover the brain for 10 to 15 min to get the desired temperature. I keep the stage temperature between -18 to -27 then

Important Note: Cutting Temperature should be 1-2ºCbelow when slide start rolling/fell down.

Jill Miotke

Dear Britanie,

I agree with Amir that it helps to trim the brain down to a region of interest if possible. I have found that one step that helps my tissue remain in the OCT compound is to replace the sucrose with OCT before placing it in the mold with fresh OCT. It is not enough to simply blot the sucrose off the tissue. I do this by placing the tissue on a glass slide and then covering it with OCT, and then carefully stirring, or simply turning the tissue over, to dilute out the sucrose. If you watch this process under a dissecting microscope you will see the Schlieren lines gradually disappear, and also be able to remove the bubbles trapped in the tissue, either with the tip of the forceps or just with time.

Good luck with your sectioning! Jill

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