TITLE: Freezing and Formalin Fixation of Tissue
AUTHORS: Jamie Allen, Maya Brewer, Elizabeth Neumann, Carrie  Romer, Danielle Gutierrez, Mark deCaestecker, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">To describe the procedure for freezing fresh tissue procured from the Cooperative Human Tissue Network.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Frozen tissue should have minimal structural damage from freezing.</div></div>

[STEPS]
?. [Documentation and Preparation]
Place a layer of dry ice in to box and add enough isopentane to make a slurry.
?. Label embedding molds and add a small amount of CMC to each one (barely cover the bottom).
?. Remove sample container from ice and document on sample sheet any information that is written on the lid.
?. [Freezing of Tissue]
Place metal weigh boat with sample on top of slurry.  Cover box with lid and allow tissue to completely freeze.
?. Write sample number and orientation on the edges of the molds (indicating inner/outer, superior/inferior).Place embedding mold with CMC on top of the slurry.
?. Check the mold in the slurry to see if it is starting to freeze.  Add tissue when the CMC starts to become opaque.  Orient the tissue so that the side that is to be cut is face down.
?. Slowly add more CMC on top of the tissue until it is covered.  Keep the lid closed over the slurry as much as possible.
?. 
?. When completely frozen, wrap in foil and label sample number with sharpie.
?. Place all samples from one patient in a labeled box and place in the.
-80 °C
?. Place disposable equipment in the Biohazard trash.  Wipe all forceps, rulers, bottles with appropriate cleansing wipes.
?. [Tissue for Fixation]
Carefully slice a small section of tissue (preferably having both cortex and medulla).  Do this in a way that you create two "bookends."  The section for fixation should be no thicker than a nickel.
?. [Tissue for Fixation]
Carefully place the piece of tissue bookend down into an embedding cassette.  Place in 10% formalin container.  After freezing the remaining sample, continue with Step 17 for fixation.
?. [Formalin Fixing and Embedding Tissue]
Allow tissue to “fix” overnight at room temperature for at least 12 hours but typically 24 hours.
?. [Formalin Fixing and Embedding Tissue]
Move tissue cassettes into 70% ethanol for 35 minutes.
?. [Formalin Fixing and Embedding Tissue]
Tissue is then embedded in more paraffin to make a block.
?. Place sample on dissection board.  Cut sample into 3-5 pieces.  These will need to be small enough to fit into the embedding mold and allow the sample to be completely covered with CMC.  Place these smaller pieces in reference to the original position and take a picture.
?. Prepare a container of 10% Formalin for preparing FFPE samples.
?. [Tissue for Fixation]
Measure and document the length, width, and height.  Place sample on metal weigh boat and document weight.  Document any other information or conditions (attachment).
?. [Formalin Fixing and Embedding Tissue]
Submerge the tissue into 90% ethanol for 35 minutes.
?. [Formalin Fixing and Embedding Tissue]
Submerge the tissue into 95% ethanol for 35 minutes.
?. [Formalin Fixing and Embedding Tissue]
Submerge the tissue into three sequential containers of 100% ethanol, each for 35 minutes.
?. [Formalin Fixing and Embedding Tissue]
Submerge the tissue into 100% xylenes for 35 minutes, three times.
?. [Formalin Fixing and Embedding Tissue]
Submerge the tissue into paraffin (was) for 60 minutes, three times.