Oct 11, 2022

Public workspaceOCT-Embedded Tissue Preparation

DOI
https://dx.doi.org/10.17504/protocols.io.81wgb6mpolpk/v1
  • Stephen Fisher1,
  • Marielena Grijalva1,
  • Rong Guo1,
  • sarahjoh 1,
  • Hieu Nguyen1,
  • John Renz2,
  • Jean G Rosario1,
  • Steven Rudich2,
  • Brian Gregory1,
  • Junhyong Kim1,
  • Kate O'Neill1
  • 1University of Pennsylvania;
  • 2Gift of Life Donor Program
Stephen Fisher
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgb6mpolpk/v1
Protocol Citation: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh , Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill 2022. OCT-Embedded Tissue Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb6mpolpk/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: August 13, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 68609
Keywords: frozen sectioning, tissue samples in optimal cutting temperature, oct tube, uncap oct tube, snap freezing, oct block, tissue, portable liquid nitrogen dewar, tissue sample, tissue section, advantage of oct, molds with the cold liquid, freezing, oct, embedding mold, thin tissue section, cold liquid, liquid nitrogen, minimal tissue processing, disrupt tissue, optimal cutting temperature, bubble formation, formation of bubble, tissue preparation
Funders Acknowledgements:
NIH
Grant ID: U54HD104392
Abstract
This protocol describes embedding of tissue samples in optimal cutting temperature (OCT) compound (Lin & Lin, 2020). Tissue embedded in OCT can then undergo frozen sectioning on a microtome-cryostat. The advantage of OCT embedding is it requires minimal tissue processing and tissue does not need to undergo fixation prior to snap freezing. OCT blocks should be stored in -80°C freezer and can yield thin tissue sections (< 5µm). Formation of bubbles in the OCT can distort and disrupt tissue. To minimize bubble formation, we recommend storing OCT upside down in a small bucket between uses. When ready for use, uncap OCT tube and dispense slowly to prevent bubbles from forming. Once tissue is in cassette surrounded by OCT, snap freezing is done. Ensure you have a portable liquid nitrogen dewar partially filled with liquid nitrogen. Place a piece of Styrofoam atop the liquid nitrogen to prevent direct contact of the embedding molds with the cold liquid.
Materials
  • Freshly dissected tissue block
  • Dry ice
  • Liquid nitrogen
  • ReagentTissue-Tek OCTFisher ScientificCatalog #14-373-65
  • Access to -80°C ultra-low freezer
  • Styrofoam (small piece)
  • Aluminum foil

Equipment
Portable liquid nitrogen dewar
NAME
Pope Scientific, Inc
BRAND
8921
SKU

Equipment
Cryomolds
NAME
Tissue-Tek
BRAND
4557
SKU
https://us.vwr.com/store/product/4639407/tissue-tek-cryomold-molds-adapters-sakura-finetek
LINK



Troubleshooting
Label biopsy molds appropriately.
Add a small amount of OCT to the bottom of each mold, ensuring no bubbles are present.
Place tissue piece in the center of the mold on top of the OCT layer.
Pour OCT slowly into the mold, ensuring the tissue piece is completely covered.
Using a small piece of aluminum foil, tightly wrap the mold.

Tip: Label the outside of the aluminum for easy retrieval later.
Place foil-wrapped mold flat in a liquid nitrogen dewar on top of the Styrofoam.
After approximately one minute, move the frozen OCT mold to the dry ice container.

Note: OCT will become opaque when frozen.
Store molds at -80℃ ultra-low freezer for Biobanking or until ready for downstream processing.