TITLE: HuBMAP: Embedding Fixed Frozen OCT Samples
AUTHORS: Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP frozen fixed specimens.</div></div>

[STEPS]
?. [Reagent Preparation]
Prepare 1x PBSPBS is required to prepare reagents needed for this procedure. Dilute 10x PBS 1:10 in deionized water to make working 1x solution.
?. [Reagent Preparation]
Prepare a 4% paraformaldehyde solution.
Paraformaldehyde is toxic. Use gloves, wear a lab coat and work in a fume hood.
?. [Reagent Preparation]
Open 2 -10mL ampules of 16% paraformaldehyde stock. Add contents to the 1x PBS in the sample cup, invert to mix.
[16% Paraformaldehyde Stock]
?. [Reagent Preparation]
Add 60ml  of 100mM PBS to a sample cup.
[PBS]
Prepare containers to accomodate number and size of tissues using  atleast a 1:20 tissue to fixative volume ratio.
?. [Tissue Preparation Procedureure]
Collect tissue specimen from the designated common coordinate framework sites. (See case processing SOP diagrams)-
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?. [Tissue Preparation Procedureure]
Place tissue into pre-labelled cassettes maintaining orientation.
?. [Tissue Preparation Procedureure]
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.
[In Fixative on Rocker]
0 Room temperature
?. [Tissue Preparation Procedureure]
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.
?. [Tissue Preparation Procedureure]
Infiltrate and cryoprotect the tissue with  30% sucrose.
[in Sucrose/PBS Mixture]
?. [Freeze the Blocks]
Prepare a pre-labeled Cryomold and fill it half way with OCT compound.
?. [Freeze the Blocks]
Remove the tissue from the cassette using foceps and quickly touch the tissue to a Kimwipe to remove  external sucrose droplet from tissue surface.
?. [Freeze the Blocks]
Place the tissue into the OCT-containing Cryomold, maintaining original tissue orientation.
?. [Freeze the Blocks]
Using forceps, push the tissue lightly to the bottom of the Cryomold to secure it.
?. [Freeze the Blocks]
Add additional OCT to cover the tissue completely and fill the Cryomold. Allow the tissue to equilibrate in the mold for ten (10) minutes.
[In Cryomold]
?. [Freeze the Blocks]
Prepare dry ice/methylbutane slurry for freezing OCT blocks.
?. [Freeze the Blocks]
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.
?. [Freeze the Blocks]
Place 1-2 inches of dry ice pellets into the bolltom of an ice bucket or styrofoam box.
?. [Freeze the Blocks]
Add enough 2-methylbutane solution to cover the dry ice by roughly 5mm .
?. [Freeze the Blocks]
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.
Enter block location in Freezer Log for future retrieval
?. [Reagent Preparation]
Prepare 30% sucrose solution
?. [Reagent Preparation]
Label containers, solution expires one week after being made. Store at 4° C if not immediately used.
?. [Reagent Preparation]
Combine 300g Sucrose with 1 liter of 1X PBS. Add stir bar and mix on a stir plate until dissolved.
[sucrose]
[ 1x PBS]
?. [Reagent Preparation]
Store at 4° C in labeled container. Check for contamination prior to use, no expiation date.
?. [Tissue Preparation Procedureure]
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
?. [Tissue Preparation Procedureure]
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]
Place lid on freezing chamber and allow methylbutane to chill. Chamber is ready when fog dissipates and bottom of bucket becomes visible.