Feb 24, 2020

Public workspaceHuBMAP: Embedding Fresh Frozen OCT Samples

DOI
dx.doi.org/10.17504/protocols.io.bcwsixee
  • 1university of Florida;
  • 2University of Florida
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Franchesca Farris
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DOI: dx.doi.org/10.17504/protocols.io.bcwsixee
Protocol Citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fresh Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bcwsixee
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 24, 2020
Last Modified: February 24, 2020
Protocol Integer ID: 33458
Abstract
The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP fresh frozen specimens.
Guidelines
  • Managers and supervisors - are responsible for making sure that technicians are properly trained and equipment and facility are maintained in good working order.
  • Laboratory personnel - are responsible for reading and understanding this SOP and related documents and to perform these tasks in accordance with the SOPs.
Materials
MATERIALS
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
ReagentTissue Tek Cryomold (25mmx20mmx5mm)Catalog #25608-916
Reagent2-MethylbutaneFisher ScientificCatalog #O3551-4
ReagentIce / Dry Ice Bucket (EVA Foam)Fisher ScientificCatalog #03-395-152
STEP MATERIALS
ReagentTissue Tek Cryomold (25mmx20mmx5mm)Catalog #25608-916
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
Protocol materials
ReagentTissue Tek Cryomold (25mmx20mmx5mm)Catalog #25608-916
Reagent2-MethylbutaneFisher ScientificCatalog #O3551-4
ReagentIce / Dry Ice Bucket (EVA Foam)Fisher ScientificCatalog #03-395-152
ReagentTissue Tek Cryomold (25mmx20mmx5mm)Catalog #25608-916
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
ReagentTissue Tek Cryomold (25mmx20mmx5mm)Catalog #25608-916
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
Safety warnings
Avoid direct skin contact with dry ice.
Methylbutane should be collectedd as hazardous waste.
Before start
  • Ensure you have proper scalpel blades, forceps, and your personal preference of gauzes/wipes.


  • Embedding can be a messy process, to protect your clothes it is best to wear a lab coat or apron.


  • Gloves are required when working with human specimens.
Collect tissue specimen from the designated common coordinate framework sites.
(See case processing SOP diagrams)

Document
Appendix for Spleen SOP
NAME

Appendix for Spleen SOP

CREATED BY
Marda Jorgensen

Document
SOP Appendix for Spleen
NAME

SOP Appendix for Spleen

CREATED BY
Marda Jorgensen

Document
SOP Appendix for Thymus
NAME

SOP Appendix for Thymus

CREATED BY
Marda Jorgensen

Prepare a pre-labeled Cryomold
ReagentTissue Tek Cryomold (25mmx20mmx5mm)Catalog #25608-916

and fill it half way with OCT compound.

ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930

Gently blot tissue piece on dry gauze to remove any excess moisture.
Place the tissue into the OCT-containing Cryomold, maintaining tissue orientation.
Using forceps, push the tissue lightly to the bottom of the Cryomold to secure it.
Prepare dry ice/methylbutane slurry for freezing OCT blocks.
Place 1-2 inches of dry ice pellets into the bolltom of an ice bucket or styrofoam box.
Add enough 2-methylbutane solution to cover the dry ice by roughly 5mm.
Place lid on freezing chamber and allow methylbutane to chill.
Chamber is ready when fog dissipates and bottom of bucket becomes visible.
Fill the mold with OCT, covering tissue completely.
Avoid bubbles.


Freeze the tissue in the Cryomold by resting it on the surface of the methylbutane slurry.
Use long handled forceps to transfer the mold onto the slurry.
As the OCT inside the Cryomold begins to freeze, lightly push the tissue into the bottom of the mold one last time.


When the OCT Cryomold is thoroughly frozen and opaque, wrap the mold containing the tissue in a pre-labeled aluminum foil square and store at -80° C in a freezer rack box.

Note
Enter block location if Freezer Log for future retrieval.