Histological and Immunohistochemical Analysis of Mouse Intestine

The excised colon from each mouse was opened longitudinally, “swiss rolled”, and fixed for 24 h in 10% (w/v) buffered formalin at room temperature. All tissues were embedded in paraffin, and 5-μm thick sections were stained with Haematoxylin and Eosin (H&E). Unstained sections were deparaffinised in xylene and rehydrated in a graded series of ethanol (100–70% v/v) finishing in dH₂O. Antigen retrieval was achieved by boiling slides in citrate buffer (ThermoFisher Scientific, AP-9003-500) or EDTA-Tris (1 mM EDTA, 10 mM Tris pH 9) for 15 min, followed by incubation in 3% (v/v) H₂O₂ to inhibit endogenous peroxidases. Sections were blocked in 5% (v/v) goat serum and incubated with primary antibodies against Bcl-G (clone 2E11, [9 (link)]), BrdU (BD Biosciences, BD555627), Ki67 (Cell Signaling, CST 9129), Caspase-3 (Cell Signaling, CST 9664), CD45 (BD Biosciences, BD553076), or p53 (Leica Biosystems, P53-CM5P-L) overnight at 4 °C in a humidified chamber. Sections were rinsed, then incubated in HRP-conjugated biotinylated secondary antibodies for 1 h at room temperature, after which HRP (Vector Laboratories) was detected using a DAB Peroxidase kit (Dako, K346811-2). All sections were counterstained with Haematoxylin. The extent of positive staining per mm [2 (link)] of tissue was quantified using ImageJ software.