Animal Perfusion Fixation for Mice
Prepare Solution
PBS (Phosphate-Buffered Saline)
PBS is the “base” for the following solutions (such as 4% PFA, 15% sucrose, etc.)
1x PBS (1L) Formula:
| Reagent | Amount |
|---|---|
| NaCl | 8.006 g |
| KCl | 0.2 g |
| Na2HPO4 | 1.44 g |
| KH2PO4 | 0.24 g |
The 1x PBS solution can be store in Room Temperature.
4% PFA (Paraformaldehyde)
PFA should be dissolved in 1x PBS solution.
1. Formula
The PFA solution is used to fix the animal tissue, it is organic. The solution should be dissolve in 1x PBS solution.
The 4% is weight per volume (m/v). For example, for 4g PFA powder should be dissolved in every 100 mL of solution.
2. [IMPORTANT] Conditions for preparing this solution
Safety:
- dThe Paraformaldehyde decomposes slowly in WATER to form toxic and flammable Formaldehyde gas. Therefore, the solution should be prepared in fume hood.
Temperature:
- It is better to heat the solution to 60 ℃ when preparing it.
- However, room temperature is also OK but the dissolution may be slower.
Accelerate its dissolution：
- 1M NaOH can be added when dissolving PFA, this step can accelerate the dissolution. (It may take 30-40 mins to completely dissolved under 60℃, but 50-60 mins under room temperature.)
- No NaOH is also OK, but the dissolution will be much slower (Should dissolve overnight).
pH adjustment:
- After dissolution, the pH of the solution should be adjusted to 7.2−7.6.
Storage:
- For 1-2 weeks, store PFA solution at room temperature.
- For a few weeks (up to one month), store PFA solution at 4℃.
- For long term storage (up to a year) at -20℃.
15% and 30% Sucrose solution
The sucrose should be dissolved in 1x PBS.
15% and 30% Sucrose in 1x PBS is used for the cryoprotection of mice brains post perfusion, which can help prevent ice crystal formation in tissues when water freezes and expands.
The 15% and 30% is also weight per volume (w/v). For example, 15g sucrose should be dissolved in every 100 mL of solution.