Paxgene-Fixed Cryo-Embedded Tissue Processing Protocol

Paxgene-Fixed Cryo-Embedded Tissue Processing Protocol

Materials

Reagents and solutions

  1. PAXgene tissue fixative
  2. PAXgene tissue stabilizer
  3. Ethanol (100%, molecular biology grade)
  4. Molecular-grade water (RNase/DNase-free)
  5. PBS Tablets
  6. EDTA (0.5 M), pH 8.0, RNase-free

Glassware and Consumables

  1. 5ml Centrifuge Tubes
  2. 5mL Serilogical pipettes or p5000 pipette tips
  3. 6-Well Plates
  4. Tissue spatulas: LevGo® 17251 STERILE Spatula

Equipment

  1. Shaker/rotating plate in cold room or 4 ºC fridge
  2. Fume hood
  3. Drying oven for paraffin embedding
  4. Flammable-safe refrigerator

Procedure

Old Protocol with pictures here

1. Fixation

  1. Wear PPE (lab coat, gloves, eye covering)
  2. Prepare fixative tubes: transfer 4 mL of PAXgene tissue fixative from a tissue fixative container (they come in 50 mL plastic jars) to a 5 mL screw-cap tube
  3. Clip live coral directly into 4 mL of PAXgene (5 mL tube)
  4. Fix 24hr at 4 ºC, gently shaking

2. Post-fixation wash (replace fixative with stabilizer)

  1. Fume hood with proper PPE
  2. Add specified amount of ethanol to PAXgene tissue stabilizer and keep at 4 ºC
  3. Prepare and label waste containers: 1 bottle and one bag for contaminated solids
  4. Remove fixative into PAXgene fixative + stabilizer waste bottle
  5. Replace with 4 mL of cold (4ºC) stabilizer. Close tube.
  6. Invert tube 3X and then discard the stabilizer into same waste bottle.
  7. Replace with fresh stabilizer and store tubes in 4ºC Flammable Fridge.
    1. Label box as containing tissue in PAXgene tissue stabilizer (flammable!)

3. Prepare PBS to store at 4ºC

  1. PBS (DNAse/RNAse-free): 10 1X PBS tablets to 1 L of RNAse/DNAse-free molecular-grade water
  2. Chill EDTA - put a 50 mL aliquot at 4ºC per six-well plate at a time
    1. Main bottle is supposed to stay at room temperature to avoid precipitation

4. Rehydrate tissue

  • Goal: to remove excess stabilizer (contains ethanol) before decalcification (EDTA and ethanol form a precipitate)
  1. Transfer tissue to DNAse/RNAse-free PBS in 6-well plate at 4 ºC
  2. Shake gently for 15 minutes to wash tissue. Can alternatively change the PBS 2x for 3 5 minute washes.

5. Decalcification

  1. Replace PBS with cold 0.5 M EDTA (pH 8.0)
  2. Seal plate with parafilm, shake gently at 4 ºC overnight-5 days (depending on sample)
  3. Replace EDTA daily or every 48hr until skeleton is dissolved.

6. Prepare sucrose solutions

  1. 15% sucrose: Add 7.5 g sucrose to 50 mL DNAse/RNAse-free PBS in a 50mL falcon tube.
    • Aliquot into 15 mL falcon tubes for each sample. Store at 4 ºC.
  2. 15% sucrose: Add 15 g sucrose to 50 mL DNAse/RNAse-free PBS.
    • Aliquot into 15 mL falcon tubes for each sample. Store at 4 ºC.

7. PBS rinse

  1. Replace EDTA in plate with PBS gently without disturbing tissue
  2. Gently shake for 5 minutes at 4ºC
  3. Repeat 2x more for 3 total rinses

8. Cryoprotection

  1. Transfer each tissue to an individually labelled 15 mL tube containing 15 % sucrose
  2. Incubate 10 minutes or until tissue sinks at 4 ºC
    • Often the tissue sinks immediately
  3. Transfer each to labelled 15 mL tube containing 30 % sucrose
    1. Incubate until tissue sinks, usually overnight

9. Embedding Protocol here

  1. Embed on powdered dry ice and store at -80 ºC until sectioning
    1. Make sure to cool forceps on dry ice and OCT to 4ºC
    2. Make sure to fully dry tissue before embedding
    3. Let tissue sit in OCT for 2 minutes before freezing, to let it fully soak in
    4. Tightly wrapped in aluminum foil cleaned with Ethanol and RNAse away, then put in whirlpak and stored at - 80 ºC in a plastic box.
    5. Cryomold, alumnium foil labelled with frag number, two wrapped molds per one labelled whirlpak per frag.

10. Sectioning and LCM, details to come

Section , prototocl refined here and finalized here

Table for test run

Day Date Step Solution Temperature Duration Start Time End Time Status
Prep Day 0 Wed, 9/10 Wash PBS 4 ºC (Cold room) 15 10:00 10:15 Done
Prep Day 0 Wed, 9/10 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 1 Thu, 9/11 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 2 Fri, 9/12 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 3 Sat, 9/13 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 4 Sun, 9/14 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 4 Mon, 9/15 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 5 Wed, 9/17 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 6 Wed, 9/17 Rinse PBS 4 ºC (Cold room) 5 11:30 11:35 Done
Prep Day 6 Wed, 9/17 Rinse PBS 4 ºC (Cold room) 5 11:35 11:40 Done
Prep Day 6 Wed, 9/17 Rinse PBS 4 ºC (Cold room) 5 11:40 11:45 Done
Prep Day 6 Wed, 9/17 Cryoprotection 15% sucrose 4 ºC (Cold room) 10 11:45 12:45 Done
Prep Day 6 Wed, 9/17 Cryoprotection 30% sucrose 4 ºC (Cold room) overnight 09:15    
Embedding Thu, 9/18 Embedding OCT On dry ice        
Written on September 10, 2025