Preparation of 4% paraformaldehyde solution for transcardial perfusions and histology
Alejandra Rangel, Rain Kwan, Asheeta Prasad
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.
Attachments
Steps
Solution preparation
Weigh 40g PFA.
Add to 500mL H2O in beaker with flea.
Heat solution to 60°C - 65°C on the heat plate with stirrer (use lowest possible heat setting).
Add 1-2 pellets of NaOH to dissolve PFA powder.
Allow solution to cool to Room temperature.
Filter dissolved PFA using ceramic vacuum filtration system into a conical flask.
Mix PFA solution with 120mL Buffer A and 380mL 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.
Label solution according to GHS guidelines.
Store at 4°C.
Wash hands with soap and water once finished and before leaving the laboratory.
Spills
The following general procedures can be used for spills involving small volumes (mL size) of dilute solutions of formaldehyde (e.g.100mL fixative solution of 4% formaldehyde) contained within a fume hood:
Wear appropriate personal protective equipment(PPE).
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> 1L, 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
Formaldehyde is very water soluble and will therefore affect mucous membranes.
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
0h 15m 0s. - Contact a first aider for assistance.
- If there is severe irritation, refer to the University medical centre.
