Feb 24, 2020

Public workspaceHuBMAP: Embedding Fixed Frozen OCT Samples

DOI
dx.doi.org/10.17504/protocols.io.basniede
  • 1University of Florida
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Marda Jorgensen
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DOI: dx.doi.org/10.17504/protocols.io.basniede
Protocol Citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fixed Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.basniede
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: December 20, 2019
Last Modified: April 02, 2020
Protocol Integer ID: 31278
Abstract
The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP frozen fixed 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
ReagentKimWipesFischer Scientific
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
Reagent16% ParaformaldehydeFisher ScientificCatalog #15710
ReagentD-Sucrose (Molecular Biology) Fisher BioReagentsFisher ScientificCatalog #BP220-1
ReagentPBS Phosphate Buffered Saline 10X Solution Fisher BioReagentsFisher ScientificCatalog #BP399-1
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® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
ReagentPBS Phosphate Buffered Saline 10X Solution Fisher BioReagentsFisher ScientificCatalog #BP399-1
16% Paraformaldehyde
PBS 10x Solution
D-Sucrose
OCT
Tissue Tek Cryomold
Methylbutane
Dry Ice
Ice Bucket
Protocol materials
Reagent16% ParaformaldehydeFisher ScientificCatalog #15710
ReagentD-Sucrose (Molecular Biology) Fisher BioReagentsFisher ScientificCatalog #BP220-1
ReagentPBS Phosphate Buffered Saline 10X Solution Fisher BioReagentsFisher ScientificCatalog #BP399-1
ReagentPBS Phosphate Buffered Saline 10X Solution Fisher BioReagentsFisher ScientificCatalog #BP399-1
ReagentKimWipesFischer Scientific
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
ReagentIce / Dry Ice Bucket (EVA Foam)Fisher ScientificCatalog #03-395-152
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
ReagentPBS Phosphate Buffered Saline 10X Solution Fisher BioReagentsFisher ScientificCatalog #BP399-1
ReagentTissue-Tek® O.C.T. Compound, Sakura® FinetekVWR International (Avantor)Catalog #25608-930
Safety warnings
  • Use physical safety precautions when working with sharps (disposable blades).
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 highly suggested to protect your fingers from the spectrum of hot and cold one encounters during the process.
Reagent Preparation
Reagent Preparation
1h
1h

Prepare 1x PBS

PBS is required to prepare reagents needed for this procedure. Dilute 10x PBS 1:10 in deionized water to make working 1x solution.
ReagentPBS Phosphate Buffered Saline 10X Solution Fisher BioReagentsFisher ScientificCatalog #BP399-1


Prepare a 4% paraformaldehyde solution.
Safety information
Paraformaldehyde is toxic. Use gloves, wear a lab coat and work in a fume hood.



Add 60ml of 100mM PBS to a sample cup.
Amount60 mL PBS


Note
Prepare containers to accomodate number and size of tissues using
atleast a 1:20 tissue to fixative volume ratio.


Open 2 -10mL ampules of 16% paraformaldehyde stock.
Add contents to the 1x PBS in the sample cup, invert to mix.

Amount20 mL 16% Paraformaldehyde Stock

Label containers, solution expires one week after being made. Store at 4° C if not immediately used.




Prepare 30% sucrose solution
Combine 300g Sucrose with 1 liter of 1X PBS. Add stir bar and mix on a stir plate until dissolved.
Amount300 g sucrose
Amount1 L 1x PBS

Store at 4° C in labeled container. Check for contamination prior to use, no expiation date.
Tissue Preparation Procedureure
Tissue Preparation Procedureure
3d 8h
3d 8h
Collect tissue specimen from the designated common coordinate framework sites.
(See case processing SOP diagrams)



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

Document
SOP Appendix for Lymph Node
NAME

SOP Appendix for Lymph Node

CREATED BY
Marda Jorgensen


-
30m
Place tissue into pre-labelled cassettes maintaining orientation.




15m
Transfer the tissue into a 120mL specimen container, pre-filled with 80mL freshly prepared fixative (4% paraformaldehyde in PBS).

Fix specimen for 24 hours at room temperature under mild agitation using a rocker.

Duration24:00:00 In Fixative on Rocker TemperatureRoom temperature

1d
Drain fixative (collect as hazardous waste) and wash the tissue three (3) times for 10 minutes each in 1X PBS at a volume that generously covers.
Invert several times during this process, or return to rocking platform.
Duration00:30:00


1h
Infiltrate and cryoprotect the tissue with 30% sucrose.
Temperature4 °C in Sucrose/PBS Mixture Duration72:00:00

2d
Combine equal volumes of 30% sucrose and 1x PBS to form 15% Sucrose. Replace PBS wash with 15% sucrose and place at 4° C for 8 hours
Drain 15% sucrose and replace with 30% sucrose. Equillibrate tissue 4° C for at least 48 hours.
Tissue is stable in sucrose for at least one week.
Freeze the Blocks
Freeze the Blocks
30m
30m
Prepare a pre-labeled Cryomold

Equipment
Cryomold®
NAME
Histology
TYPE
Tissue Tek®
BRAND
25608-916
SKU
https://us.vwr.com/store/product/4639407/tissue-tek-cryomold-molds-adapters-sakura-finetek
LINK
25mm x 20 mm x 5mm
SPECIFICATIONS

and fill it half way with OCT compound.

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



1m
Remove the tissue from the cassette using foceps and quickly touch the tissue to a Kimwipe to remove external sucrose droplet from tissue surface.
1m
Place the tissue into the OCT-containing Cryomold, maintaining original tissue orientation.
1m
Using forceps, push the tissue lightly to the bottom of the Cryomold to secure it.
1m
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.
Freeze the tissue in the Cryomold by resting it on the surface of the methylbutane slurry.
As the OCT inside the Cryomold begins to freeze, lightly push the tissue into the bottom of the mold one last time.
Add additional OCT to cover the tissue completely and fill the Cryomold.
Allow the tissue to equilibrate in the mold for ten (10) minutes.
Duration00:10:00 In Cryomold

20m
When the OCT Cryomold is completely 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 in Freezer Log for future retrieval