Organs are cryoprotected by immersion in 30% sucrose solution (in phosphate buffer) overnight. Sections are cut using a cryostat (10 ^m) or a freezing microtome (100-200 ^m) and washed 3X in washing buffer, at 20 min intervals. Incubation in X-gal reaction solution is best carried out overnight in a 37°C oven (see Note 7). To ensure that the sections do not dry out, an adequate volume of reaction solution should be added (e.g., 1 mL for a 35-mm Petri dish). Gentle shaking on a rocking platform is optional; this ensures even penetration of the substrate. An intense blue reaction product should be evident by 12 h. Sections are then rinsed in wash buffer and mounted on slides for dehydration in graded ethanols, cleared, and cover-slipped using Permount. The reaction product is stable in mounted sections. A light bluish background stain is sometimes seen in certain tissues (see Note 8).
Was this article helpful?