Animal harvesting for immunohistochemical analysis:
Male C57BL6/J mice with or without renal stenosis were sacrificed via
CO2 asphyxiation followed by cervical dislocation.
Contralateral (right) renal arteries were excised and placed in 4%
paraformaldehyde in PBS for 24 hours then placed in 100% ethanol for
processing. Tissues were embedded in paraffin and sectioned at 8 μm
thickness. Immunohistochemical analysis was performed as previously
described (Durgin, et al., 2019). Tissue sections were deparaffinized
with xylenes and rehydrated by sequentially decreased concentrations of
ethanol (100%-70%) followed by deionized distilled water.
Heat-mediated antigen retrieval was then performed using citric
acid-buffer (Vector Laboratories, H-3300) for 20 minutes, then sections
cooled for 30 minutes at 4ºC. Sections were then blocked in 10% horse
serum (Sigma H1138) in PBS (MilliporeSigma, H1270) at room temperature
for 1 hour. Primary antibodies (See Table 1) for sGCβ (Cayman Chemical,
160897, 1:100) and von Willebrand Factor (VWF; Abcam, ab11713, 1:250)
were incubated on sections in PBS containing 10% horse serum overnight
at 4ºC in a humidity chamber. One section per slide was stained with
rabbit (Vector Laboratories, I-1000) IgG control to match corresponding
sGCβ antibody concentration. Tissue sections were washed thrice, 5
minutes each with PBS. Sections were then incubated in PBS containing
10% horse serum with smooth muscle α-actin (ACTA2) primary antibody
pre-conjugated to FITC fluorophore (MilliporeSigma, F3777 clone 1A4,
1:500), 4′,6-diamidino-2-phenylindole (DAPI, D3571, Thermo Fisher
Scientific, 1:100) and secondary antibodies (See Table 1) donkey
anti-rabbit AlexaFluor 594 (Invitrogen, A-21207, 1:250) and donkey
anti-sheep AlexaFluor 647 (Invitrogen, A-21447, 1:250) for 1 hour at
room temperature in a humidity chamber. Tissue sections were then washed
thrice in PBS for 5 minutes before being mounted on coverslips using
Prolong Gold Antifade mounting medium with DAPI reagent (Invitrogen,
P36931). Immunohistochemistry staining of renal arteries were imaged
using a Nikon A1 Confocal Laser Microscope at the University of
Pittsburgh Center for Biological Imaging. Images were taken with 40X
objective magnification with 1024 x 1024 pixel resolution. Increments
for Z-stacks of 1 μm were applied for stained and IgG controls. In
ImageJ, a region of interest was drawn around ACTA2+ areas representing
the smooth muscle cell tunica media then superimposed on sGCβ images for
quantification of medial smooth muscle sGCβ expression.