Preparation of fresh paraformaldehyde for mouse perfusion and tissue fixation
Thomas Hnasko
Published: 2023-12-12 DOI: 10.17504/protocols.io.6qpvr3qbbvmk/v1
Abstract
Hnasko lab - preparation of fresh PFA for ms perfusion and tissue fixation
Steps
1.
- For
1Lof 4% paraformaldehyde, warm up700mLof ddH2O in a glass beaker using microwave until40-60°C.
2.
- Place on heated stir plate in a chemical fume hood, add a magnetic stir bar and start stirring and insert thermometer into solution.
3.
- Weigh
40gof granular paraformaldehyde (PFA, EMS, cat# 19210).
4.
- Add PFA to the heated ddH2O solution and place on a stir plate in a chemical fume hood. Insert thermometer. Stir and heat to approximately
60°C(paraformaldehyde flash point = 71°C). Avoid overheating.
5.
- The powder will not immediately dissolve into solution. Slowly raise the pH by adding about
1mLNaOH 10N until the solution clears.
6.
- Once the paraformaldehyde is dissolved, add
100mL10X PBS (Fisher bioreagents, cat# BP399-1).
7.
- Cool solution to
Room temperaturebefore adjusting pH, can place on ice to speed cooling.
8.
- Adjust pH to ~
7.4with small amounts of 12N HCl.
9.
- Adjust the volume of the solution to
1Lwith ddH2O.
10.
- Filter the solution using a vacuum filtration flask (ThermoScientific, cat# 567-0020). Label and store in sealed container at
4°C.
