Preparing OCT blocks of kidney tissue (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Steve Potter

Keywords

[‘Laser Capture Microdissection’]

Subjects

[‘Isolation, purification and separation’, ‘Gene expression analysis’]

Release Date

2017-08-07

Abstract

Preparing OCT blocks of kidney tissue protocol by the Potter Group

Procedure

1. Dissect out kidneys rapidly and store briefly (up to 20 min or so) in ice cold PBS.
2. Process through OCT only as many kidneys as you plan to freeze in one block at a time. The OCT is hyperosmotic and tends to suck the water out of the tissue it touches, ruining the edges of the kidney, where a lot of interesting stuff is happening. So faster is better. But, at the same time, you need to totally rinse the PBS away, because it can interfere with the sectioning.
3. Place kidneys in OCT in a 60 mm plate cooled with ice. Use OCT that has been pre-cooled on ice for at least ten minutes.
4. Quickly mix kidneys in with OCT, then transfer carefully to a new 60 mm plate with cold OCT, quickly mix again, and place in a tinfoil mold with cold OCT covering the bottom.
5. Cover kidneys with additional OCT and position kidneys near each other in mold, in central position, not too near the top.
6. Immediately freeze in 2-methylbutane isopentan that is in a pyrex beaker resting in liquid nitrogen. The isopentan should be frozen. Hold the tinfoil mold with forceps to keep vertical and gently move to keep from freezing in the isopentan and to improve thermal contact.
7. When the OCT is completely frozen place the mold in dry ice, and then store long term in liquid nitrogen freezer.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium