X-gal Staining Procedure

Materials
Fixative
2% Paraformaldehyde (in PBS)

Staining solution
5 mM potassium ferricyanide,
5 mM potassium ferrocyanide,
2 mM MgCI,
0.01% sodium deoxycholate
0.02% Nonidet P-40 (NP-40)
in PBS.

X-gal Stock
40 mg/mL X-Gal (5 -Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)
in dimethylformamide
store at -20°C in small aliquots.

X-gal Mix (make fresh just before use)
10 mL Staining solution
0.25 mL X-gal Stock (final = 1 mg/mL).
Be sure that X-Gal is well dissolved before use otherwise crystals may form in the staining solution

Procedure
1) Fix cells on ice for the following amount of times: 
    15':  Cells, small organs (pituitary, adrenal, thyroid), E8 embryos.
    30':  Organ slices (2-3 mm), E10 embryos.
    60':  Organ slices (4 mm), E12 embryos.
    90':  Whole organs, E14 embryos.

2) Cut organs on a Vibratome in ice cold PBS to a thickness of 0.5 mm. 

3) Transfer tissues to X-gal mix in small dishes or multi-well plates.  Incubate at 37°C with gentle agitation until desired level of staining is achieved.  Stain longer if histologic sections will be cut.

4) Place tissue slices on a small square of lens paper and transfer to histology tissue cassettes. The lens paper helps to keep the specimen from curling up during paraffin processing.