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.