X-Gal Staining

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(See also the protocol in “Current Protocols in Molecular Biology”)

 

1. Wash cells 2 X in Tris / PBS.

2. Fix in 4% paraformaldehyde in PBS; 15 minutes at Room Temp;

    5 mL for 10 cm, 2 mL for 6 cm.

3. Wash 3 X in PBS / Tris (2nd wash 10 min).

4. Stain in X-Gal Solution; ~3 mL for 10 cm; stain several hours to overnight at 37C.

 

Solutions

1 X PBS:

 

4 % paraformaldehyde: dilute 20 % paraformaldehyde into 1 X PBS

 

Coloid developer (X-Gal Solution):

            X-Gal              1 mg / mL        (Stock: 50 mg / mL in DMF)

            K4Fe(CN)6     5 mM               (Stock 100 mM)

            K3Fe(CN)6     5 mM               (Stock 100 mM)

            MgCl2             1 mM               (Stock 1 M)

 

            In 1 X PBS / Tris

 

100 mM KxFe(CN)6:

            K4Fe(CN)6     4.2 g                (100 mM)

            K3Fe(CN)6     3.3 g                (100 mM)

 

            Add 100 mL 1 X PBS / Tris

 

20 % paraformaldehyde (pfa):

            pfa                               100 g

            hot (60 – 65C) water   300 mL

            20 X PBS                    25 mL

            Bring to 450 mL with DI H2O

            Add NaOH until clears (pH 7-8)

            Cool to 4C

            Filter with 0.45 um filter and aliquot