Intestinal tissue was fixed in 10% formalin or 4% paraformaldehyde overnight at 4°C, embedded in paraffin (H&E and in situ) or cold optimum cutting temperature embedding medium (H&E). Tissues were processed for paraffin-embedding and H&E staining by the CCHMC Digestive Health Center Pathology Core. In situ hybridization was performed in the lab following the published protocol (Gregorieff and Clevers, 2010 ). Both H&E and in situ images were captured using Nikon scientific microscope AX series.
Intestinal tissue was fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose, embedded in OCT compound, and sectioned while frozen at 12 μm.
Images were acquired on a Nikon A1R GaAsP Inverted Confocal Microscope, with signal intensity optimized for control samples and the same settings (laser power/gain/offset) used for KO and rescue tissues. p-histone H3+ cell numbers were counted (20 crypts per mouse in 3 mice) to get the average p-histone H3+ cells per crypt. Results are expressed as the mean ± standard deviation, and significance calculated by Student’s t test (p < 0.05 was considered significant).
Intestinal tissue was fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose, embedded in OCT compound, and sectioned while frozen at 12 μm.
Images were acquired on a Nikon A1R GaAsP Inverted Confocal Microscope, with signal intensity optimized for control samples and the same settings (laser power/gain/offset) used for KO and rescue tissues. p-histone H3+ cell numbers were counted (20 crypts per mouse in 3 mice) to get the average p-histone H3+ cells per crypt. Results are expressed as the mean ± standard deviation, and significance calculated by Student’s t test (p < 0.05 was considered significant).