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The vast majority of IHC/ICC procedures employ fixation of tissues and cells using formaldehyde-based fixatives. The protocol below describes the technique for generating a 4% formaldehyde solution in PBS. The most effective fixative must be determined experimentally.
Caution: Formaldehyde is toxic. Please read the MSDS before working with this chemical. Gloves and safety glasses should be worn and solutions made inside a fume hood.
Please read the protocol in its entirety before starting.
• Filter units
• Glassware and stir bar (dedicated for formaldehyde solution) • Gloves and eye protection
• Hot plate with magnetic stirrer
• Thermometer
• Ventilated hood
• Deionized H2O
• HCl (Dilute)
• NaOH (1 N)
• Paraformaldehyde powder
• 1X PBS: 0.145 M NaCl, 0.0027 M KCl, 0.0081 M Na2HPO4, 0.0015 M KH2PO4, pH 7.4
1. For 1 L of 4% Formaldehyde, add 800 mL of 1X PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil.
2. Add 40 g of paraformaldehyde powder to the heated PBS solution.
3. The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH dropwise from a pipette until the solution clears.
4. Once the paraformaldehyde is dissolved, the solution should be cooled and filtered.
5. Adjust the volume of the solution to 1 L with 1X PBS.
6. Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9.
7. The solution can be aliquoted and frozen or stored at 2-8 °C for up to one month.