Time required for fixation and decalcification of mouse tissues

Hao Chen/Ryan Rountree (version 1.0; Jan 16, 04)

	Whole mount embryos	Embryo limbs	Early postnatal limbs (<14 days)	Adult limbs (>14 days)
4% PFA	15-20 min, cut the embryo into half, another few minutes to make the total 45 min	45 min	3 hours	4 hours
Decalcification	no	no	no	2-4 days

Decalcification of adult mouse limbs (> 14 days)

Hao Chen/Ryan Rountree Jan 16, 04

Cal-EX (for just histology)

1. Dissect skin away from sample as much as possible, and immediately place into 15ml tube containing prechilled PBS on ice (kill one animal at a time, and if skin of the fingers cannπt be removed, at least cut all the way down the length of finger through the dorsal skin).

2. Fix in 10 Volume of 4% PFA for 4 hours.

3. Wash with PBS for 15 min x 3.

4. Put on Cal-EX ((Fisher Scientific, CS510-1D; a HCl + chealating agents ≠ make a waste container for it) for 1-2 hour at RT (test with needle or pipette tip for softness).\

5. Wash with PBS for 15 min x 3.

6. Refix with 4% PFA for 1 hours.

7. Wash with PBS for 15 min x 3.

8. Put into 15% sucrose in PBS for 1 hour and 30% sucrose in PBS O/N.

EDTA (to preserve lacZ activity)

1. Dissect skin away from sample as much as possible, and immediately place into 15ml tube containing prechilled PBS on ice (kill one animal at a time, and if skin of the fingers cannπt be removed, at least cut all the way down the length of finger through the dorsal skin).

2. Fix in 10 Volume of 4% PFA for 4 hours.

3. Wash with PBS for 15 min x 3.

4. Put on EDTA (0.5 M, PH 7.4) for 4 days.

5. Wash with PBS for 15 min x 3.

6. Put into sucrose: 15% for 1 hour and 30% O/N.

Note: all procedures are performed at 4∞C unless noted, the samples are then embedded in O.C.T. and section at12mm, CT=-22ö, OT=-26ö, knife angle=3ö. You can also decalcify and section old skeletal preps with Cal-EX. Just stir the tissue in a beaker full of water O/N to fully rinse out glycerol.