Immunocytochemical analysis of hypertrophy:
RPGSMCs were cultured on a single 24x50 mm cover glass (VWR, 16004-322)
to approximately 90% confluency and then serum starved in DMEM/F12 for
48 hours. Media was then gently aspirated by hand and the cover glass
washed with PBS containing 0.1% Triton X-100 for 5 minutes. Cells were
then fixed in 4% paraformaldehyde in PBS for 30 minutes at room
temperature and then gently washed twice with PBS containing 0.1%
Triton X-100. RPGSMCs blocking was carried out in PBS with 10% horse
serum for 1 hour at room temperature. Primary antibody incubation for
rabbit-sGCβ and sheep-vWF (See Table 1) incubation was carried out in
PBS with 10% horse serum overnight at 4ºC in a humidified chamber.
Cells were then gently washed thrice with PBS for 5 min each time before
being incubated in either primary ACTA2 antibody conjugated to FITC
fluorophore or AlexaFluor donkey anti-rabbit secondary antibody (See
Table 1) for 1 hour at room temperature in PBS containing 10% horse
serum. Cells were gently washed twice for 5 min in PBS before applying
Prolong Gold Antifade mounting medium with DAPI reagent (Invitrogen,
P36931). Immunocytochemistry images of RPGSMCs were taken using a Leica
DM1000 microscope at 40x objective with 2X zoom applied and cell area
was quantified using ImageJ software.