X-Gal (Synonyms: BCIG, X-β-Gal, 5-bromo-4-chloro-3-indolyl-β-D-Galactopyranoside)

Protocol for X-gal storage configuration:

X-gal storage configuration: 20mg/ml storage solution prepared by dissolving X-gal with dimethylformamide. Weigh 20g X-gal, add 1L dimethylformamide (DMF), dissolve and store in a bottle, which is wrapped in aluminum foil to prevent damage due to light, and should be stored at -20℃. X-gal solution does not require filtration to remove bacteria.

Protocol for X-gal Staining on Adult Mouse Brain Sections ^[1]:

1. The mouse is perfused with PBS for 5 minutes, followed by 4% PFA/PBS for another 5 minutes.
2. The mouse brain is dissected out and post-fixed with 4% PFA/PBS for 4 hours at 4°C.
3. The fixed brain is first washed with PBS three times. Following this, it is incubated with 20% sucrose overnight (or until the samples sink), and then with 30% sucrose overnight. This process helps in cryoprotection and preparation of the brain tissue for cryosectioning.
4. The brain is mounted in O.C.T. compound on a cryomold and stored at -80℃ until use.
5. 40 μm cyro-section is made using cryostat and mounted on the slide-glass.
6. The slide is washed once with a 150 ml staining buffer for 10 minutes at room temperature.
7. The slide is incubated with 1 mg/ml X-gal in staining buffer containing 5 mM Potassium Ferricyanide and 5 mM Potassium Ferrocyanide at 37℃ until color develops. This typically takes 3 hours to overnight. Ensure samples don't dry out and use 100-200 μl X-gal solution per slide.
8. Stained sample is washed with PBS three times (5 min each).
9. The slide is dehydrated using a series of ethanol solutions (50%, 75%, 90%, 100%, 2 minutes each).
10. The slide is mounted with permount and can be stored at room temperature. The sample is now ready for imaging at higher magnification.

Reference:

[1]. Kokubu H, Lim J. X-gal Staining on Adult Mouse Brain Sections. Bio Protoc. 2014;4(5):e1064. doi: 10.21769/bioprotoc.1064. Epub 2014 Mar 5. PMID: 27390760; PMCID: PMC4932900.