TITLE: Preparation of 4% paraformaldehyde solution for transcardial perfusions and histology
AUTHORS: Alejandra Rangel, Rain Kwan, Asheeta Prasad

[DESCRIPTION]
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.

[STEPS]
SECTION: Spills
13. The following general procedures can be used for spills involving small volumes (mL size) of dilute solutions of formaldehyde (e.g.100 mL fixative solution of 4% formaldehyde) contained within a fume hood:
SECTION: Spills
13.1. Wear appropriate personal protective equipment(PPE).
 

SECTION: Spills
13.2. Remove any ignition source from the spill area (particularly if the solution has been stabilised with methanol).
SECTION: Spills
13.3. If possible, boost ventilation to the area.
SECTION: Spills
13.4. Absorb any residual solution with absorbent.
SECTION: Spills
13.5. Clean spill area with detergent and water.
SECTION: Spills
13.6. Dry decontaminated spill area with paper towels.
SECTION: Spills
13.7. Dispose of any absorbent material along with other chemical waste through the university hazardous waste program.
SECTION: Spills
14. If a large volume of concentrated formaldehyde solution is spilled (e.g> 1 L, 37% formaldehyde) or if the spill occurs outside a chemical fume hood or ventilated area, immediately:
SECTION: Spills
14.1. Alert others working in the area.
SECTION: Spills
14.2. Evacuate the area.
SECTION: Spills
14.3. Restrict the access to the area.
SECTION: Spills
14.4. 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.
SECTION: Exposure
15. Formaldehyde is very water soluble and will therefore affect mucous membranes.
 

SECTION: Exposure
16. The following first aid procedure should be promptly undertaken;
SECTION: Exposure
16.1. 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.
SECTION: Exposure
16.2. Skin or eye exposure:
Remove contaminated clothing / contact lenses.
Immediately flush with tepid water for at least 15 min.
Contact a first aider for assistance.
If there is severe irritation, refer to the University medical centre.

SECTION: Solution preparation
1. Weigh 40 g PFA.
SECTION: Solution preparation
7. Mix PFA solution with 120 mL Buffer A and 380 mL Buffer B (recipes below).
SECTION: Solution preparation
6. Filter dissolved PFA using ceramic vacuum filtration system into a conical flask.
SECTION: Solution preparation
5. Allow solution to cool to Room temperature.

SECTION: Solution preparation
4. Add 1-2 pellets of NaOH to dissolve PFA powder.
SECTION: Solution preparation
3. Heat solution to 60 °C - 65 °C on the heat plate with stirrer (use lowest possible heat setting).
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2. Add to 500 mL H2O in beaker with flea.
SECTION: Solution preparation
8. Adjust pH solution to 7.4 using 2N HCl. Only use the pH meter designated for PFA preparation.
SECTION: Solution preparation
12. Wash hands with soap and water once finished and before leaving the laboratory.
SECTION: Solution preparation
11. Store at 4 °C.
SECTION: Solution preparation
10. Label solution according to GHS guidelines.
SECTION: Solution preparation
9. Transfer solution into 1 L Schott bottle.