Hi!
Please, can anyone explain the mechanism whereby the reporter gene, lacZ, is used in staining a piece of tissue for subsequent visualization of the product of the gene it is connected to? (I know that in the blue/white screening, it cleaves beta-gal and produces blue color, but how about in tissue itself???)
Thank you so much.
Cat

When doing stainings looking at LacZ gene expression (Beta Galactosidase) you would usually use a staining solution that contains potassium ferricyanide, potassium ferrocyanide and magnesium chloride (5mM, 5mM and 2mM, respectively).

X-Gal is the substrate that is added to this staining solution. Our stock of X-Gal was at a concentration of 1mg/mL in DMF. I used to use 100 microlitres of X-Gal per 4mL of staining solution. Pre-warm it and add it to the cells or tissue that had been fixed with 2% Formaldehyde, 0.2% Glutaraldehyde in PBS.

The conversion reaction that takes place when Beta galactosidase uses X-Gal as substrate produces a purple (i think) precipitate. This usually appears blue in tissues and because it is a precipitate, it does not leak out of the cells over time (as GFP might).