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LacZ staining protocol--beta galactosidase (lacZ) Staining

This procedure describes how to process samples for lacZ staining. Beta galactosidase is an enzyme that hydrolizes beta galactosides. The cleavage of Xgal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside) results in a dark blue precipitate. A nuclear localized lacZ transgene can be used to mark transgene expressing cells unambiguously (endogenous enzyme activity is cytosolic). If desired, antibody staining can be carried out for cytosolic proteins (see Brinkmeier et al. ). Thick specimens, such as late stage mouse embryos, can be cleared by treatment with (see Turkay et al.). Background staining can be reduced by increasing the pH of the phosphate buffers used to process samples. For a review see the chapter by Saunders.

lacZ Staining Procedure for Cells and Mouse Embryos:
1. Rinse cells (or mouse embryos) once in phosphate buffer pH 7.3 at room temperature.
2. Fix cells for 5 minutes (embryos for 15-30 minutes, depending on size) at room temp.
3. Wash cells 3 times for 5 minutes (15 minutes for embryos) at room temperature.
4. Stain cells (embryos) 4 hours to overnight at 37*C depending on level of LacZ activity.
5. After staining, pour off stain, replace with wash buffer.
6. Store samples at 4*C. Staining will intensify in wash buffer at 4*C.

Notes on Staining:
The procedure doesn't have background problems in most cell types. With embryos, background staining is seen in the yolk sac of day 10 embryos and on. In addition, a thin stripe of staining is observed in the hindbrain of day 12 embryos. If background is a problem, then try increasing the pH of the phosphate buffer. Staining procedure works at pH 8.5.

Staining Procedure for Cryostat Sections:
1. Collect tissues or embryos and place in isopentane at -30 to -45*C for 30 seconds.
2. Store frozen samples at -70*C.
3. Section embryo.
4. Fix sections for 5 minutes- use glutaraldehyde fix.
5. Wash 3 times for 5 minutes.
6. Stain sections overnight at 37*C.
7. After staining, pour off and save stain, replace with wash buffer. Can counterstain with neutral red, dehydrate in ethanol and xylene and coverslip.
8. Store samples at 4*C. Staining will intensify in wash buffer at 4*C


Materials and Reagents

0.1 M phosphate buffer, pH 7.3:
115 ml 0.1 M sodium phosphate, monobasic (6.9 g in 500 ml H2O)
385 ml 0.1 M sodium phosphate, dibasic (14.2 g in 500 ml H2O)
500 ml total

Fix Solution - Always prepare fresh:
4 ml 25% glutaraldehyde
2.5 ml 100 mM EGTA, pH 7.3
0.4 ml 1 M MgCl2
173.1 ml double distilled H2O
20.0 ml 10x PBS
200.0 ml total

Wash Buffer:
0.4 ml 1 M MgCl2
2.0 ml 1% deoxycholate (may be omitted)
2.0 ml 2% NP-40
195.6 ml 0.1 M sodium phosphate, pH 7.3
200.0 ml total

X-gal stock:
250 mg X-gal (5-bromo-4-chloro-3-indolyl-b -galactopyranoside) (Sigma B4252)
10 ml dimethyl formamid (Sigma D4551)

X-gal stain - Make fresh:
2.0 ml 25 mg/ml X-gal stock
0.106 g potassium ferrocyanide (Sigma P-9387)
0.082 g potassium ferricyanide (Sigma P-8131)
48.0 ml wash buffer
50.0 ml total