Lac-Z Detection in Tail Biopsies

1. 2mm tail biopsies are collected in 1X PBS (see recipes below) in a 1.5ml microfuge tube. Be sure to include a known +/+ (lac-Z negative) biopsy as a control.

2. Replace 1X PBS solution over biopsy with 2.5% Glutaraldehyde diluted in SpM buffer (see recipes below).

3. Pre-warm microwave by placing 300ml H2O in a beaker in microwave and heating on "HI-Power" setting for 30 sec. Remove from microwave.

4. Microwave biopsy 7-10 sec. with microfuge lids open.

5. Wash biopsy 3 times in 1X PBS.

6. Replace final PBS wash with 200-500ul staining medium (see recipes below); enough to cover tissue completely.

7. Incubate @ 37 C 1-3 hrs (alternatively the incubation can proceed overnight at room temperature).

8. By 15 min. of incubation blue color should be visible at the exposed biopsy surface (where the tail was cut ).

9. Stop reaction by replacing staining solution with 1X PBS. These may be stored at 4 C for several weeks. During this time color deposition will begin to leach into solution.

Reagents:

1 X Phosphate Buffered Saline (PBS)

0.2g                 KCl

0.2g                 KH2 PO4,

1.15g               Na2HPO4

8.0g                NaCl____

to 1 liter with ddiH2O

1X SpM

2mM                  CaCl2

2mM                  MgCl2

0.3%                 Glucose

0.3%                Fructose

made in 1X Phosphate Buffered Saline

Staining Medium

5mM                  Potassium Ferricyanide

5mM                  Potassium Ferrocyanide

1 mg/ml            X-gal (in dimethyl sulfoxide) final concentration

0.1M                Hepes (pH 7.4)                                                  

made in 1X SpM

Adapted from:
Murti, JR and Schimenti, JC. "Microwave-Accelerated Fixation and Lac-Z Activity Staining of Testicular Cells in Transgenic Mice". Analytical Biochemistry :198, pp. 92-96 (1991).