New PBS-PFA Recipe

The PBS-PFA tested on 9/4/2012 seemed to work fine, but for the future we are changing to a more common variant of Sorensen's phosphate buffer, based on recipes from  UC Berkley Biological Imaging Resources and Wikipedia—Phosphate Buffered Saline, and adapted for use with anhydrous Na2HPO4 (Berkley used 7●H2O, and Wikipedia 2●H2O)

PBS, pH 7.4: 

For 1 liter of 20X Stock use:

KCl                                         4 g

NaCl                                   160 g

Na2HPO4 (anhydrous)          22.9  g

(Sodium Phosphate Dibasic)

KH2PO4                                  4 g

(Potassium Phosphate Monobasic)

For 1 liter of PBS-Working, add 50 ml 20x stock to 950 ml DI

4% PFA in PBS, pH 7.4:

• For 1 liter of 4% Paraformaldehyde (PFA) in PBS, add 40 g PFA per liter of working PBS 
• For 4 liters of 4% PFA in PBS, add 200 ml 20x PBS Stock to 3800 ml DI.
• Heat to 60 - 70º C (Do Not Exceed 70ºC) and add 160 g PFA. Add NaOH (0.5 g/l) if needed to get PFA in solution. 
• Filter the PFA, with coffee filter, when cool enough to touch
• Move filtered PFA to 4º C overnight, or use salted-ice bath if perfusing the same day 
• Measure pH of cold PFA (4 - 8º C) and adjust pH to 7.4 with a few drops of HCl (12 N) or NaOH (1 to 5 N) if needed. 
• Prepare Sucrose Postfix (10 to 20% Sucrose in cold, pH'd 4% PFA). 
• Postfix for one animal = 11.5 g sucrose in 75 ml PFA (= 15%). For 3 animals = 35 g sucrose in 225 ml PFA; and postfix for 4 animals = 46 g sucrose in 300 ml PFA.

Perfusion Protocol:

Perfuse animals with normal saline (9g NaCl/liter) for about 5 min at a pump setting of 1.1 (= 40 ml/min) and then switch to PFA. When rigor sets in (or about a minute, whichever comes first), reduce pump speed to 0.8 (= 25 ml/min) for 20 min. Total volume of PFA perfused should be just over 500 ml. Remove the brain and place in cold sucrose postfix for 18 to 24 hr.