Feb 09, 2023

Public workspaceCryosectioning fresh frozen tissues for multimodal imaging

DOI
dx.doi.org/10.17504/protocols.io.e6nvwjmjzlmk/v1
  • 1Columbia University;
  • 2Pennsylvania State University
Presha Rajbhandari
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.e6nvwjmjzlmk/v1
Protocol Citation: Presha Rajbhandari, Taruna Neelakantan, Hua Tian, Brent R Stockwell 2023. Cryosectioning fresh frozen tissues for multimodal imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjmjzlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 09, 2023
Last Modified: February 09, 2023
Protocol Integer ID: 76738
Abstract
This protocol describes sectioning for fresh frozen tissues for multimodal imaging including Hematoxylin & Eosin staining, DESI imaging mass spectrometry, SIMS imaging mass spectrometry and RNAscope imaging.
Guidelines
Upon finishing cryosectioning, remove the blade and dispose in sharps container before cleaning the cryostat.
Discard tissue remnants in biohazard bag.
Materials
Cryostat, Leica- CM 3050S
Accu-Edge Microtome Blades, Sakura- 4689
Anti roll glass, Leica 14047742497
Clean the cryostat surface, anti-roll blade and forceps with ethanol before sectioning.
Place the tissue block inside the cryostat at -20°C for 30 mins for equilibration. Also, place the slides and substrates inside the cryostat for cooling.
Set the chamber temperature of the cryostat to -20C and object temperature to -18C to -20C
Place the blade in place and lock it.
Trim the tissue block at 30-50µm thickness to obtain a flat surface.
Mount the tissue block on the chuck using water (no OCT) and adjust the angle of the chuck as needed.
Position the anti-roll bar appropriately so that it doesn’t scrape the tissue (if placed too far) or roll off of the block (if placed too near).
Gently section the tissue at 8µm thickness using an anti-roll bar and place the consecutive sections on individual substrates. Adjust and flatten with a fine tipped brush if necessary. For H&E, DESI and RNAscope imaging, tissue sections are placed on regular or positively charged microscope slides and on gold coated 2.5cm2 substrate for SIMS-imaging.
Thaw mount the sections on the target using heat from the finger under the slide.
Store at -80C until use.