ROSA26 : Lac-Z Detection in Tail Biopsies
- 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.
- Replace 1X PBS solution over biopsy with 2.5% Glutaraldehyde diluted in SpM buffer (see recipes below).
- Pre-warm microwave by placing 300ml H2O in a beaker in microwave and heating on "HI-Power" setting for 30 sec. Remove from microwave.
- Microwave biopsy 7-10 sec. with microfuge lids open.
- Wash biopsy 3 times in 1X PBS.
- Replace final PBS wash with 200-500ul staining medium (see recipes below); enough to cover tissue completely.
- Incubate @ 37 C 1-3 hrs (alternatively the incubation can proceed overnight at room temperature).
- By 15 min. of incubation blue color should be visible at the exposed biopsy surface (where the tail was cut ).
- 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.
1 X Phosphate Buffered Saline (PBS)
0.2g KH2 PO4,
to 1 liter with ddiH2O
made in 1X Phosphate Buffered Saline
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
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).