Aug 22, 2022

Public workspacePreparation of 4% paraformaldehyde solution for transcardial perfusions and histology

DOI
dx.doi.org/10.17504/protocols.io.yxmvmndobg3p/v1
  • Alejandra Rangel1,
  • Rain Kwan1,
  • Asheeta Prasad1
  • 1The University of Sydney
Benjamin Trist
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DOI: dx.doi.org/10.17504/protocols.io.yxmvmndobg3p/v1
Protocol Citation: Alejandra Rangel, Rain Kwan, Asheeta Prasad 2022. Preparation of 4% paraformaldehyde solution for transcardial perfusions and histology. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmndobg3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68560
Keywords: Paraformaldehyde, Tissue fixation, Perfusion, Histology, ASAPCRN
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes the safe preparation of 4% paraformaldehyde solution for use in tissue fixation in preparation for histology and immunohistochemistry. Paraformaldehyde is extremely toxic and must be handled with great care. We therefore also provide advice for the safe handling and disposal of this solution.
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Materials
Equipment:

  • Weighing spatulas
  • Amount2 L heat resistant (up to 500 degrees) glass beaker
  • Hot plate with stirrer
  • Magnetic stirrer bar
  • Ceramic vacuum filtration system with conical flask
  • Amount2 L Schott bottle

Consumables:

  • Whatman Filter Paper
  • Formaldehyde spills kit

Key reagents:

  • ReagentParaformaldehyde (prilled 95%)Sigma AldrichCatalog #441244
  • Sodium hydroxide pellets (Thermo #CPC-AJA482)
  • Hydrochloric acid

Solutions:

  • Buffer A: Concentration0.2 Molarity (M) NaH2PO4
1. Amount13.8 g in Amount500 mL dH2O
  • Buffer B: Concentration0.2 Molarity (M) Na2HPO4
1. Amount14.194 g in Amount500 mL dH2O
Safety warnings
  1. This procedure will take place entirely inside the fume hood.
  2. Place designated PFA scale and hot plate and stirrer inside the fume hood, these should not be used for any other purpose.
  3. Adequate PPE including double nitrile gloves, lab gown, safety glasses should be worn at all times, with gloves replaced regularly and breaks taken at least every 15 minutes throughout.
  4. Place PFA in progress sign on exterior of room door, close the door.
Solution preparation
Solution preparation
Weigh Amount40 g PFA.
Add to Amount500 mL H2O in beaker with flea.
Pipetting
Heat solution to Temperature60 °C - Temperature65 °C on the heat plate with stirrer (use lowest possible heat setting).
Add 1-2 pellets of NaOH to dissolve PFA powder.
Pipetting
Allow solution to cool to TemperatureRoom temperature .

Filter dissolved PFA using ceramic vacuum filtration system into a conical flask.
Mix PFA solution with Amount120 mL Buffer A and Amount380 mL Buffer B (recipes below).
Adjust pH solution to 7.4 using 2N HCl. Only use the pH meter designated for PFA preparation.
Transfer solution into 1 L Schott bottle.
Pipetting
Label solution according to GHS guidelines.
Store at Temperature4 °C .
Wash hands with soap and water once finished and before leaving the laboratory.
Wash
Spills
Spills
The following general procedures can be used for spills involving small volumes (mL size) of dilute solutions of formaldehyde (e.g.Amount100 mL fixative solution of 4% formaldehyde) contained within a fume hood:
Wear appropriate personal protective equipment(PPE).
Note
This should include a reusable respirator if there is not sufficient local area ventilation.

Remove any ignition source from the spill area (particularly if the solution has been stabilised with methanol).
If possible, boost ventilation to the area.
Absorb any residual solution with absorbent.
Clean spill area with detergent and water.
Dry decontaminated spill area with paper towels.
Dispose of any absorbent material along with other chemical waste through the university hazardous waste program.
If a large volume of concentrated formaldehyde solution is spilled (e.g> Amount1 L , 37% formaldehyde) or if the spill occurs outside a chemical fume hood or ventilated area, immediately:
Alert others working in the area.
Evacuate the area.
Restrict the access to the area.
Contact Laboratory Supervisor for use of formaldehyde spills kit containing a urea-based formaldehyde neutralizer (e.g. Spill X-FPTM) which will chemically react with formaldehyde to form a polymer product that is no longer volatile and much less hazardous. These formaldehyde spill neutralizers are commercially available. Locate the spills kit within easy access to where formaldehyde is handled or stored.
Exposure
Exposure
15m
15m
Formaldehyde is very water soluble and will therefore affect mucous membranes.
Note
Symptoms of acute exposure can result in eye and throat irritation, coughing, wheezing, chest tightness, bronchitis, laryngitis, clouding of the cornea and loss of vision.

The following first aid procedure should be promptly undertaken;
Exposure via inhalation.
  • Immediately move the person to fresh air.
  • Contact a first aider and/or refer the person to the University medical center.
  • If the person is having respiratory difficulties, call an ambulance.
Skin or eye exposure:
  • Remove contaminated clothing / contact lenses.
  • Immediately flush with tepid water for at least Duration00:15:00 .
  • Contact a first aider for assistance.
  • If there is severe irritation, refer to the University medical centre.

15m