β-galactosidase detection in embryos and tissues using X-Gal

1.  Dissect embryos or tissue PBS as rapidly as possible.

2.  Fix in 4% paraformaldehyde in PBS supplemented with xx% triton X-100 xx time at 4C with rocking

Timing of fixation and triton concentration depend on the embryo stage:

For E10.5 embryos, fix 1h in 4% PFA, E12.5 embryo 1h 4% PFA with 0.1% triton, E14.5 2h 4% PFA with 0.2% triton, E17.5 3h 4% PFA with 0.5% triton. Put individual embryos in 2ml tubes or small vials 

3.  Rinse 2-3 times over 20 min with PBS with rocking at 4C

4.  Incubate in X-Gal solution overnight at 37C:

^aUltrapure X-gal SIGMA : 15520-018

5.  After incubation, rinse 2-3 times over 20-30 min with PBS at 4C

6.  Postfix in 4% paraformaldehyde several hours to overnight at 4C

7.  Store in PBS at 4 °C (could add 0.1% Na-azide) till imaging

Reagents: 

* 16% Paraformaldehyde (formaldehyde) aqueous solution: (EMS, ref 15710 ,10x10 ml) single use aliquots

* 10% triton (prepare fresh, i.e. 1ml triton + 9ml of PBS, warm at 37C to dissolve, store at 4C for maximum two weeks)

* Prepare PFA + triton fixative before use

NOTE: if X-gal staining is to be done on cryo-sections:

- fix embryos the time indicated above

- wash in PBS 2h to overnight at 4C

- equilibrate with sucrose 30% in PBS

- embed and freeze in OCT

- do cryosections (12-18 microns)

- let dry cryosections 10 min RT

- wash cryosections in PBS

- incubate cryosections in a small slide holder containing X-Gal at 37C Overnight in the dark

- wash with PBS 3x 5 min RT

- perform further stainings if needed