Diaminobenzidine tetrahydrochloride dab

The mice were anesthetized with isoflurane and euthanized at defined time points or when moribund. Colon tissues were then removed and fixed in 10% Formalin overnight, prior to embedding in paraffin. One hr prior to euthanasia, BrdU (0.04 g/kg) was injected i.p. into the mice. Sections of formalin-fixed, paraffin-embedded mouse colon tissues were utilized for immunohistochemical analysis using the following primary antibodies: mouse anti-BrdU (1∶300), mouse anti-β-catenin (1∶800), rabbit anti-phospho-S6 (1∶100), rabbit anti-phospho-mTOR (1∶100) and rabbit anti-Sox9 (1∶1000). Antigen retrieval was performed by heating the sections in microwave in Antigen Retrieval Citra Solution (Biogenex, Fremont, CA) for 15 minutes prior to application of the primary antibodies. Appropriate secondary antibodies were used and the reaction was visualized using diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories), and cell nuclei were visualized by counterstaining with hematoxylin. BrdU staining was quantified by counting the number of positive and negative cells in at least 20 crypts and the percentage of positive cells per crypt was then calculated. The crypts were then divided into 3 equal parts (top, middle, and bottom) and the quantification was performed again.

Paraffin-embedded liver tissue sections were routinely de-paraffinized with xylene and a graded series of ethanol. Some tissue sections were stained with hematoxylin–eosin (HE) for simple morphological examination. For immunostaining on paraffin-embedded liver tissues, paraffin blocks were sliced into 5 μm sections, deparaffinized with xylene, and rehydrated with decreasing concentrations of ethanol in water. Antigen retrieval was achieved by incubation for 20 min in hot (95 °C) sodium citrate buffer (pH 6.0) and 20 min of cooling at room temperature. Endogenous peroxidases were quenched by incubation in 3% hydrogen peroxide for 20 min. Sections were washed with PBS. Primary antibodies were applied for 60 min at room temperature in a humidified chamber. After rinsing the slides in PBS, they were incubated in secondary antibody for 1 h at room temperature. After washing with PBS, slides were incubated with Vectastain ABC (Vector Laboratories) for 30 min. After washing with PBS, color development was achieved by applying diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories) for two to 5 min. Images were acquired and the signal intensity was quantified using a Keyence BZ-X700 microscope.