Measuring cell proliferation in the colon epithelium
Mice (n=6, males) were provided sildenafil or malonyl-sildenafilad libitum in the drinking water dissolved at a concentration that delivered 9 mg/kg daily. Mice were weighed daily, and after at least 5 days of continuous consumption they were sacrificed, and the colon was dissected and processed for quantitation of proliferation by flow cytometry (proximal region) and by IHC (distal region). For flow cytometry, the epithelium was enriched by isolating crypts as described previously (Islam et al., 2022), followed by ethanol fixation and staining with phycoerythrin-conjugated anti-mouse Ki-67 antibodies according to the manufacturer instructions (Biolegend, San Diego, CA). Flow cytometry was carried out using a BD Accuri C6 flow cytometer, and data was analyzed using FCS Express 7 software (De Novo Software, Pasadena, CA). Processing the distal colon tissues for histology and immunohistochemistry was carried out as previously described (Wang et al., 2012). Briefly, tissues were fixed with 10% formaldehyde, embedded in paraffin blocks, and sectioned by the Augusta University histology core. The tissues were probed using antibodies to PCNA (1:100; Cell Signaling, Danvers, MA). Visualization of PCNA antibodies was done using the ImmunoCruz ABC kit (Santa Cruz Biotechnology, Dallas, TX, USA). At least 10 different regions of colon containing approximately 8 crypts per region were counted for each mouse. For both the flow-cytometer and immunohistochemistry approaches, each group (control, sildenafil, malonyl-sildenafil) contained 5 mice (male) and the experiment was reproduced in 2 independent replicates.