Protocol for Cryoembedding PAXgene-fixed Tissues in OCT Media for Cryosectioning (for FISH, Immunofluorescence, and LCM)

Example Protocols and Video

See Video from 10X genomics: Video

PAXgene cryoembedding protocol

The goal of this protocol is to embed tissue to create tissue sections that preserve structural integrity and maintain the integrity of RNA and proteins for downstream imaging and RNA extraction

Therefore, all of these steps should be performed with gloves and in a clean workspace

Supplies

1. OCT Embedding Medium
2. Cryomolds
3. Forceps
4. Dry Ice
5. Mortar and Pestle for making powdered dry ice
   • or: small dewar of liquid nitrogen (no more than 50-100 mL needed)
6. Optional: Sucrose

Preparation

1. Clean bench with clean paper towels (spray solution, wipe down) in the following order:
   • 10% bleach solution
   • DI water
   • 70% ethanol
   • RNAse cleaner (spray bottle)
2. Sterilize forceps in same way
3. Cool OCT on ice (not dry ice) for at least 30 mins
4. Cool sterilized forceps on dry ice for at least 30 mins
5. Label cryomold with sample information and orientation of tissue
   • The OCT will freeze and become opaque, so a diagram is useful to know the orientation of the tissue for sectioning
   • Place cryomold in labelled bag on dry ice to keep cool until ready
6. If using powdered dry ice instead of liquid nitrogen, crush 2-3 pellets of dry ice using a cleaned mortar and pestle

Input material:

1. Start with PAXgene fixed tissue in PAXgene tissue stabilizer that has just finished decalcification or that has been kept at -80 ºC since being placed in stabilizer (protocol)
   • Keep tissue at -80 or on dry ice until it is placed in embedding mold
2. Perform “cryoprotection” by transfering from PAXgene stabilizer to a > 10mL volume of 30% sucrose solution at 4ºC for several hours or overnight, once the tissue sinks you can continue processing. If it never sinks, overnight should be OK.
   • 
   • Alternatively you can place the tissue in a 15% sucrose solution at 4ºC until tissue sinks then to a 30% solution for several hours or overnight.
   • Read online people do this in PBS! Might be better than what I have been doing (RNAse/DNAse-Free H2O)

Embedding

1. Fill labelled cryomold with cooled OCT, being careful to not make any bubbles
   • If bubbles form, push to side of block with clean pipette tip
2. Using sterilized and cooled forceps, transfer tissue from PAXgene stabilizer or sucrose to a piece of parafilm and then dab with the corner of a kim wipe to remove excess liquid then immediately place into OCT in cryomold in pre-determined orientation
3. Cover tissue with more OCT so no tissue is exposed to air
   • Let tissue equilibriate to OCT for 5-10 mins? Could do in cold room
4. Immediaetely place cryomold on powdered dry ice so all outside surfaces of the mold are touching powdered dry ice until OCT block is completely opaque
   • OR: Place (do not submerge, LN2 should not touch OCT, the top should remain exposed) in liquid nitrogen until OCT block is completely opaque
     • This is apparently not as good for the tissue, preferred is dry ice or dry ice-100% ethanol slurry
5. Once embedded tissue block is opaque, wrap in aluminum foil and then seal in labelled zip-lock bag or whirlpack and immediately transfer (on dry ice) to -80 freezer

Example protocols:

• https://www.protocols.io/view/embedding-and-freezing-fresh-human-tissue-in-oct-u-bp2l64o55vqe/v3
• https://med.nyu.edu/research/scientific-cores-shared-resources/sites/default/files/frozen-tissue-preparation-with-sucrose.pdf