August 1, 2016

X-gal Staining Procedure


M. Fero — 3/24/04 — adapted from P. Soriano


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


  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.

Tags: Fero-Histology, Fero-Lab-Protocols