Formalin-Fixed Paraffin Embedded Tissue Processing Protocol

Formalin-Fixed Paraffin Embedded Tissue Processing Protocol

Materials

Reagents and solutions

  1. Filtered (0.2 µm), UV-sterilized seawater or sewater-like buffer (mPBS)
  2. 37% Formaldehyde Solution, ACS/Histology grade (Macron Chemicals Cat. # MK-5016-02)
  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
  7. Xylenes (store and dispose in glass only)
  8. Parrafin wax: Paraplast X-TRA®

Glassware and Consumables

  1. Glass bottles
  2. 5ml Centrifuge Tubes
  3. 5mL Serilogical pipettes or p5000 pipette tips
  4. 6-Well Plates
  5. Tissue spatulas: LevGo® 17251 STERILE Spatula
  6. Coplin Staining Jar
  7. Glass Petri Dishes
  8. Histology casettes: System III Biopsy Cassette
  9. Base molds: Stainless Steel Base Mold, 24 x 24 x 5mm

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

1. Prepare 4% formalin in seawater

  1. Filter seawater (in my case taken from the Putnam lab wetlab Blue Tanks) with a 0.2 uM filter into an autoclaved glass bottle
    1. I use a 30 mL syringe with an attached filter
    2. FSW hereforth means UV-sterilized filtered seawater
  2. Close bottle tightly and UV sterilize for 30 minutes
  3. In fume hood with PPE: Add 32.4 mL of formaldehyde to 267.6 mL FSW
  4. Aliquot into 5mL tubes and store at 4ºC

2. Fixation

  1. Wear PPE (lab coat, gloves, eye covering)
  2. Clip live coral directly into 4 mL of formalin (5 mL tube)
  3. Fix 24hr at 4 ºC, gently shaking

3. Post-fixation wash (replace fixative with 70% ethanol)

  1. Fume hood with proper PPE
  2. Prepare 70% ethanol solution (with RNAse/DNAse free water) and keep at 4 ºC
  3. Prepare and label waste containers: 1 bottle and one bag for contaminated solids
  4. Remove fixative into formalin waste
  5. Replace with 4 mL of cold (4ºC) 70% ethanol. Close tube.
  6. Invert tube 3X and then discard the ethanol into same waste bottle.
  7. Replace with fresh ethanol and store tubes in 4ºC Flammable Fridge.
    1. Label box as containing tissue in 70% ethanol (flammable!)

4. Prepare PBS and ethanol solutions to store at 4ºC

  1. PBS (DNAse/RNAse-free): 10 1X PBS tablets to 1 L of RNAse/DNAse-free molecular-grade water
  2. Ethanol in PBS
    1. 50% ethanol: 25 mL 100% ethanol, 25 mL PBS
    2. 30% ethanol: 15 mL 100% ethanol, 35 mL PBS
    3. 10% ethanol: 5 mL 100% ethanol, 45 mL PBS
  3. 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

5. Rehydrate tissue

  • Goal: to remove excess ethanol before decalcification (EDTA and ethanol form a precipitate)
  1. Sequentially replace solutions at 4 ºC with gentle shaking:
    1. 70% ethanol -> 50% ethanol (5 min)
    2. 50% ethanol -> 30% ethanol (5 min)
    3. 30% ethanol -> 10% ethanol (5 min)
    4. 10% ethanol -> RNAse/DNAse-free PBS (5 min)
    5. PBS -> fresh PBS (5 min)
  2. Transfer tissue to PBS in 6-well plate (5 min)

6. 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.

7. PBS rinse

  1. Replace EDTA in plate with PBS gently without disturbing tissue
  2. Gently shake for 5-15 minutes at 4ºC
  3. Repeat 2x more for 3 total rinses

8. Ethanol dehydration (can be done in 6-well plate or in beakers)

  1. Prepare solutions in DNAse/RNAse-free Water (not PBS) and store at 4ºC
    1. 50% ethanol: 25 mL 100% ethanol, 25 mL water
    2. 70% ethanol: 35 mL 100% ethanol, 15 mL water
    3. 80% ethanol: 40 mL 100% ethanol, 10 mL water
    4. 95% ethanol: 47.5 mL 100% ethanol, 2.5 mL water
  2. Move tissue to histology cassettes to move through beakers, label with pencil/alcohol-resistant ink
    1. Alt: keep tissue in 6-well plate and replace the solution in the wells.
    2. Alt: move tissue wihthout cassette using tissue spatula
  3. Transfer tissue through the following solutions:
    1. 50% ethanol, 1 hr (4ºC)
    2. 70% ethanol, 1 hr (4ºC)
      1. Can pause here overnight in 5mL tube at 4 ºC
    3. 80% ethanol, 30 min (4 ºC)
    4. 95% ethanol, 30 min (4 ºC)
    5. fresh 95% ethanol, 30 min (4 ºC)
    6. 100% ethanol, 30 min (4 ºC) x 3
      1. Replace twice for 1.5 hour total

9. Clearing

  1. All steps in fume hood. Xylenes degrade plastic – only store in glass containers
    1. (Also for xylene waste)
  2. Incubate in covered container in xylene, 20 min X 3
    1. Replace xylenes twice for 1 hour total
    2. Use glass coplin jar, beaker, or glass petri dish

10. Infiltration with and Embedding in paraffin

(Below is based almost entirely on the Squid Book, 2023 MBL Symbiosis Course, written by Derrick Kamp from Spencer Nyholm lab:)

Set up oven (54 ºC):

  1. Line bottom of oven with aluminum foil to catch any drips of wax
  2. Place forceps, molds, and all tools used during embedding on an aluminim foil-lined tray in oven so they do not harden wax upon contact
  3. Fill four autoclaved 250 mL beakers halfway with paraffin wax and place on tray
  4. (Paraffin I, II, III, and IV)
  5. Turn on oven (to 54 ºC) well ahead of time (at start of dehydration) to allow wax to melt
  6. Put a foil-covered metal plate (or something?) at -20 ºC to be a solidifying plate
  7. If tissue is not already in a cassette, label a tissue casettes for each sample

Infiltration:

  1. Monitor and keep temperature constant so wax stays liquid without degrading tissue
  2. Submerge tissue in paraffin I for 45 min
  3. Submerge tissue in paraffin II for 45 min
  4. Submerge tissue in paraffin III for 45 min

Embedding:

  1. Pour a small amount of paraffin IV into embedding mold in the oven
  2. Using pre-heated forceps, move sample to paraffin mold
    1. Orient tissue to desired position
    2. This step is time sensitive, as wax will start to cool and get sticky quickly
  3. Remove lid from tissue cassette, place cassette open side up on top of tissue for labelling
  4. Fill mold with paraffin IV
  5. Place tissue mold on solidifying plate on ice amd then pop out of mold and move to 4 ºC when fully hardened
  6. Solid blocks should be stored at 4ºC and shipped to facility for processing ASAP to maintain RNA integrity

Protocol resourses/references:

  1. http://bridgeslab.sph.umich.edu/protocols/index.php/Paraffin_Embedding_of_Tissue_Samples
  2. https://www.leicabiosystems.com/us/knowledge-pathway/an-introduction-to-specimen-processing/
  3. https://kb.10xgenomics.com/hc/en-us/articles/30139708328333-What-best-practices-should-be-considered-when-preparing-FFPE-samples-for-the-Visium-HD-Spatial-Gene-Expression-assay

Table for test run

Day Date Step Solution Temperature Duration Start Time End Time Status
Prep Day 0 Sat, 8/16 Rehydration 50% ethanol 4 ºC (Cold room) 5 9:00 9:05 Done
Prep Day 0 Sat, 8/16 Rehydration 30% ethanol 4 ºC (Cold room) 5 9:10 9:15 Done
Prep Day 0 Sat, 8/16 Rehydration 10% ethanol 4 ºC (Cold room) 5 9:20 9:25 Done
Prep Day 0 Sat, 8/16 Rehydration PBS 4 ºC (Cold room) 5 9:30 9:35 Done
Prep Day 0 Sat, 8/16 Rehydration PBS 4 ºC (Cold room) 5 9:40 9:45 Done
Prep Day 0 Sat, 8/16 Rehydration PBS 4 ºC (Cold room) 5 9:50 9:55 Done
Prep Day 0 Sat, 8/16 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 1 Sun, 8/17 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 2 Mon, 8/18 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 3 Tue, 8/19 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 4 Wed, 8/20 Decalcification EDTA 4 ºC (Cold room) 24hr 10:00 10:00 Done
Prep Day 5 Thu, 8/21 Rinse PBS 4 ºC (Cold room) 15 10:00 10:15 Done
Prep Day 5 Thu, 8/21 Rinse PBS 4 ºC (Cold room) 15 10:20 10:35 Done
Prep Day 5 Thu, 8/21 Rinse PBS 4 ºC (Cold room) 15 10:40 10:55 Done
Prep Day 5 Thu, 8/21 Dehydration 50% ethanol 4 ºC (Cold room) 60 11:00 12:00 Done
Prep Day 5 Thu, 8/21 Dehydration 70% ethanol 4 ºC (Cold room) 60 12:05 13:05 Done

https://docs.google.com/spreadsheets/d/1SJMfpZj-5SD8RbZVLp7VZ-n5NsYAllHIbdNL9VNWNZM/edit?usp=sharing

To autoclave:

  • 7 beakers for xylene and paraffin
  • embedding molds
  • forceps
Day   Step Solution Temperature Duration Start Time End Time Status
Embedding Tue, 8/26 Dehydration 80% ethanol 4 ºC (Cold room) 30 8:00 8:30  
Embedding Tue, 8/26 Dehydration 95% ethanol 4 ºC (Cold room) 30 8:35 9:05  
Embedding Tue, 8/26 Dehydration 95% ethanol 4 ºC (Cold room) 30 9:10 9:40  
Embedding Tue, 8/26 Dehydration 100% ethanol 4 ºC (Cold room) 30 9:45 10:15  
Embedding Tue, 8/26 Dehydration 100% ethanol 4 ºC (Cold room) 30 10:20 10:50  
Embedding Tue, 8/26 Dehydration 100% ethanol 4 ºC (Cold room) 30 10:55 11:25  
Embedding Tue, 8/26 Clearing Xylenes RT (Fume Hood) 20 11:30 11:50  
Embedding Tue, 8/26 Clearing Xylenes RT (Fume Hood) 20 11:55 12:15  
Embedding Tue, 8/26 Clearing Xylenes RT (Fume Hood) 20 12:20 12:40  
Embedding Tue, 8/26 Infiltration Paraffin 54ºC (Drying Oven) 45 12:45 13:30  
Embedding Tue, 8/26 Infiltration Paraffin 54ºC (Drying Oven) 45 13:35 14:20  
Embedding Tue, 8/26 Infiltration Paraffin 54ºC (Drying Oven) 45 14:25 15:10  
Embedding Tue, 8/26 Embedding Paraffin 54ºC (Drying Oven) 15 15:15 15:30  
Embedding Tue, 8/26 Embedding Paraffin On ice 15 15:35 15:50  
Written on August 16, 2025