Protocol for Cryosectioning PAXgene-fixed Tissues (for FISH, Immunofluorescence, and LCM)

Starting material: Tissue fixed, stabilized, decalcified (protocol), and embedded in OCT

The goal of this protocol is 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. Microtome blades
3. Coated microscope slides
4. Cling wrap
5. Forceps
6. Paintbrushes
7. Razor Blades
8. For imaging/making slides:
   1. Hoechst Trihydrochloride, Trihydrate
   2. PAP Pen
   3. Mounting Media
   4. Cover slips
9. Box for slides
10. Slide mailers for staining/storing slides

Input material:

1. Start with fixed tissue, embedded in a block of OCT that has been kept at -80 ºC in a sealed container (large tube, wrapped in foil, in whirlpack, etc)
2. 30 minutes before sectioning, move block containing tissue to -20 ºC freezer. Keep in sealed container. Transport to cryostat on dry ice.
3. Label slides with pencil

Sectioning

1. Clean cryostat and all tools with 70% Ethanol and/or RNAse zap (depending on application)
   • Clean cryostat, microtome blade and tools with a 1:1 mixture of RNaseZap and 70% ethanol (RNaseZap/70% ethanol) and then with sterile 95% ethanol (Recipes 1-3). Wipe dry with Kimwipes. (from Roy et al 2020)
2. Use clingwrap to cover internal surfaces
3. Place all tools in cryostat and allow to equilibriate to cryostat temperature (~30 mins!, speed up by transporting on dry ice before sectioning)
4. Mount sample onto chuck by placing a drop of OCT on the chuck and then placing the demoulded block on top
   • Do so on the holders on the left side of the cryostat, these will help to quickly freeze the OCT
5. Place chuck into sample holder and tighten all screws. Place fresh microtome blade in the blade holder and make sure all clamps and levers are locked.
6. Trim off excess OCT from the outside to make the section area smaller, making sure not to cut the tissue. If you do not know where the tissue is, wait to trim OCT until you can see the face of the section.
7. “Face” the tissue by using the “trim” setting (thicker sections, ~30 uM) to reveal the desired tissue face
   • Use arrow buttons on left side of cryostat to move the block to the blade.
   • Goal is to get a nice clean section across the whole face of the block.
   • To do so, you will have to adjust the angle of the sample holder accordingly. As you trim away sections, you will notice the first try will not be nicely in the center of the block. Adjust the angle of the sample holder horizontally and vertically and keep “trimming” until you get a flat section across the whole block.
   • Always lock the wheel when adjusting things inside the cryostat and stay clear of the blade!
   • Uses brushes to remove unwanted sections and regularly clean any residue from the blade with kimwipes
8. Once desired “face” is achieved, sectioning for the slides can begin
   • A good time to move to a different section of the blade, which you should do periodically especially if you notice any issues with the sections going through
   • Turn off the “trim” setting and set the thickness for sectioning.
   • 10 uM thickness is ideal, but thicker may be easier and may have less rolling issues.
9. Adjust roll plate so sections stay flat
   • This is the hardest part!
   • If you are having issues, make sure to regularly clean the blade (carefully!) and the anti-roll plate with Ethanol and/or RNAse zap. Any nicks or residue will hamper good sectioning. Make sure to clean the backside of the blade too, which is best done by removing the blade.
   • Other possible fixes: adjust blade or sample angle (I have had success with ~3º blade angle)
     • Leica suggests to start at an angle of 0º and slowly (1º at a time) adjust up
   • If there is any bunching or ripping of the sections, adjust the temperature colder (for crumpling/bunching) or warmer (for ripping/cracking/splintering). I have had success with -15 ºC - -20ºC.
   • **See Leica cryostat manual for troubleshooting guide (page 47).
   • Try to keep all materials inside the cryostat to keep them at the desired temperature.
10. Sectioning pictures
11. Pick up sections using slide
    • Remove the anti-roll plate and gently use paintbrushes to slide sections away from the blade, pick up the section on the frosted side of the slide (or membrane side if for LCM) with a rolling motion. Warm the slide from the back with the tip of your finger behind the section to allow the section to quickly melt. Place on the cryostat metal plate (where the blade is) to refreeze.
12. Keep slides in the cryostat and transport in sealed containers (slide mailers wrapped in parafilm and then placed in zip lock bag) on dry ice
13. When done, use a razor blade or forceps to pop off the block from the chuck. To keep block for later, cover exposed tissue with OCT and allow to freeze before storing in a sealed container and transporting to -80 ºC freezer on dry ice.
14. Cleaning: remove the clingwrap from the inside, hopefully with any excess sections contained inside. Clean inside of cryostat with 70% ethanol (without pouring liquid inside), and make sure everything is as you found it.
    • Dispose of used blades in sharps bin

Quick slide staining w/ Hoescht

• Might try to get H&E stains to do morphology checks as well

• For staining, take slides out of freezer and let any condensation evaporate.

  • Optional: Remove excess OCT with forceps, should peel right off
  • Rinse slides in 1X PBS for 10 minutes to remove OCT (in 50mL falcon tube)
    • or 70 % Ethanol
  • Use pap pen to encircle section and cover with PBS, perform staining and washing steps by pipetting off the drop of liquid and replacing with staining solution
  • Hoescht staining:
    • make 1 µg/ml solution of Hoescht
      • add 0.001 g to 1 mL of RNAse/DNAse free H2O
      • wrap tube in aluminum foil to keep dark and store at -20ºC
    • Add 100 uL of the Hoescht solution to each section and allow to stain in the dark for 5 mins.
  • Wash in 1X PBS in dark
  • Remove excess liquid by aspirating and pat dry area around sample with Kimwipe
  • Air dry, covered to prevent dust from accumulating
  • Apply a few microliters of mounting media, avoiding any bubbles
    • Mounting media tips
      • Take mounting media out of the fridge ~30 mins before mounting and store upside down to try to limit bubbles forming and getting onto the tissue
  • Cover with a large coverlslip (22x50)
  • Seal with nail polish allow slides to cure overnight (4 ºC in dark).
  • Image using confocal!
    • Hoescht: excited with 410‐nm, collect at 490‐nm (bandpass)
    • Chlorophyll: 410‐nm, collect at 610‐nm (long‐pass)
    • GFP: excited with 488‐nm, collect at 533‐nm (bandpass)

Helpful vidoes and references

Roy et al 2020 Bioprotocols

• Sectioning tips:
  • Video
  • Video 2
  • Video 3
  • “Where to put hands”
  • Anti-roll plate leica manual

• Paxgene protocol

• Helpful notes about cryosectioning for LCM

• Example Immunohistochemistry Protocol for frozen tissue sections

• Notes on extracting RNA from PAXgene fixed cryo-embedded tissues
  • Paxgene recommends extracting RNA using the PAXgene Tissue RNA/miRNA Kit, handbook
    • And has the following notes about extracting using this kit for Paxgene Fixed, Cryoembedded (PFCE) Samples from sections and LCM microdissections.
    • Paxgene recommends extracting DNA using the PAXgene Tissue DNA Kit, handbook
      • And has the following notes about extracting using this kit for Paxgene Fixed, Cryoembedded (PFCE) Samples from sections and LCM microdissections.
• See my LCM protocol for an application of this

Other Species examples

Sectioning Porites compressa

Sectioning Montipora capitata, cut at -19 ºC