Solutions

A) 1M Sodium Phosphate Buffer

Solution 1                    1M sodium phosphate, monobasic, monohydrate

NaH₂PO₄·H₂O = 137.99 (Sigma S9638)

Dissolve 69 g into 500 ml of H₂O.

Dissolve slowly over low heat.

Solution 2                    1M sodium phosphate, dibasic, anhydrous

Na₂HPO₄ (anhydrous) = 142.0 (Sigma S9763-1kg)

Dissolve 56.8 g into 400 ml of dH₂O.

Add solution 1 (monobasic) into 400 ml of solution 2 (dibasic), monitoring with a pH meter.

Adjust pH to 7.4.  Takes approximately 200 ml of solution 1 to 400 ml of solution 2.  Volumes may change, so start with smaller amounts of solution 1 and gradually add to adjust pH.

Store at RT.  When precipitates form, warm to ~50^oC.

 

B) 8% Paraformaldehyde

Should be made fresh for perfusions.  OK to use old fix for dissociated cells.

Dissolve 8g of paraformaldehyde in 0.1M sodium phosphate buffer, pH7.4, in a total volume of 100 ml.

Add 250 µl of 3N NaOH.  Continue stirring and heat at <60^oC until solution becomes clear, >30 min.

Filter (use filter paper in a funnel and a side-armed flask).  Can be stored at 4^oC for a few days.

 

C) Periodate-Lysine-4% Paraformaldehyde Fixation McLean and Nakane, 1974

Good for glycoprotein antigens.

Need >70 ml / adult mouse

1. Dissolve 3.67g of Lysine HCl in 100 ml of 0.1M sodium phosphate buffer, pH7.4.
2. Prepare 8% PFA as above.
3. Immediately before use, mix B and C at a ratio of 1:1. (i.e. 100 ml of A : 100 ml of B).

For lower PFA concentrations, e.g. 3% or 2%, adjust accordingly.  (Depends on proteins to be detected)

4. Add sodium metaperiodate to 0.01M. (NaIO₄  428 mg / 200 ml).

The final concentration of the solution should be:

4% paraformaldehyde, 0.1M lysine, 0.01M NaIO₄, and 0.1 M NaP buffer

 

Perfusion method

1. Anesthetize the mouse with isoflurane in bell jar.
2. Perfuse at RT. (Can do a preperfusion with PBS – backfill line 3 seconds –remove air bubbles from the line)

Usually finish 75 ml / adult mouse in 7-10 min.

3. Let perfused mice rest on ice for 30 min – 1 hour.
4. Dissect tissues and postfix for 1-2 hours in the same fix in 30-ml poly bottles at 4^o (fixative might precipitate on ice, better in 4^oC refrigerator)
5. Rinse 4x in cold 0.2M sodium phosphate buffer, pH7.4 > 1 hour. Better to do short solution changes initially.  Can leave tissue in the last solution overnight.

For cryosections, transfer to 30% sucrose in 0.2M sodium phosphate buffer and leave until tissue sinks to the bottom.

For free-floating sections, cut within a few days and store sections in cryostorage solution at -20^oC.

 

Chemicals:

Sodium phosphate – see above

Lysine: DL-Lysine = 183.7 (Sigma L-6001) or L-Lysine HCl = 182.6 (Sigma L-6027)

Sodium metaperiodate:            NaIO₄ = 213.9              Sigma S-1878

Paraformaldehyde                    (CH₂O)_n = (30.03)_n                     EM Sciences 19200 (500g)