Bacterial beta-Galactosidase Histochemisty Bible

This is a collection of procedures and lore associated with staining mammalian tissue for bacterial beta-galactosidase. Unfortunately, I can't attribute much of the development work, since I was generally given protocols second or third-hand. I've noted cases where I haven't used the presented procedure myself. The subcellular localization of the beta-galactosidase doesn't seem to be significant for choosing a procedure or variant conditions. I've used cytoplasmic, nuclear, and axonally localized versions without problems.

Eric Mercer ( - Last modified April 25, 1995

Whole Mount X-gal Histochemistry of Transgenic Animal Tissues

This is the method I use routinely.


  1. Remove tissue or fetus and fix in fresh 4% paraformaldehye/PBS (pH 7.0-7.5) for 1 hour at 4oC.
  2. Rinse three times, for thirty minutes each, with rinse buffer (recipe given below) at room temperature.
  3. Stain between 4 and 48 hours, typically overnight. About 90% of potential staining will occur in the first 24 hours.
  4. Post-fix overnight in 10% formalin (10% formalin is fine because freshness isn't critical) at 4oC.



Tissues with background staining

Some Variations

Clearing X-gal Stained Embryos

If you've never done this, you'll be delighted with glassy pale yellow embryos with intensely blue stained tissues inside. They look like jewelry.

Methyl Salicylate

This is the method I use.
  1. Post-fix embryos in 10% formalin overnight at 4oC.
  2. Wash with distilled water twice, each for 30 minutes at room temperature.
  3. Dehydrate as follows (all at room temperature with agitation): 30 minutes in 70% ethanol, 30 minutes in 95% ethanol, two times in 100% ethanol for 30 minutes each.
  4. Transfer to 100% methyl salicylate, and agitate at room temperature (or 4oC), until the tissue clears. Clearing takes about 15 minutes for E10.5 mice, and about an hour for E15.5 fetuses.

Cedar Wood Oil

  1. Dehydrate embryos through graded ethanol steps up to 100% ethanol
  2. Place embryos in Xylene:100% ethanol (1:1) for 1 hour.
  3. Transfer embryos to high quality cedarwood oil and rock gently, 1-4 hours.
  4. Replace cedarwood oil once and rock gently overnight.

Benzyl Benzoate

  1. Dehydrate embryos through graded steps into 100% methanol.
  2. Transfer embryos into benzyl benzoate:benzyl alcohol (1:1).

X-Gal Histochemisty of Culture Cells

This procedure is faster and simpler than the procedure for whole tissues. An important difference is the addition of gluteraldehyde to the fix, to more firmly attach cells to the culture plate.

Reference: EMBO 5:3133 (1986), modified by Jaques Peschon.

  1. Aspirate off medium and rinse once with PBS (pH 7.3)
  2. Fix 5 minutes on ice.
  3. Rinse three times with PBS (1-4 minutes each).
  4. Stain at 37oC, overnight.
  5. Rinse once with PBS, and store in PBS at 4oC


Notes 1