Novared chromagen

Macaque tissue was fixed in 10% formalin and paraffin embedded. Five μm sections were stained with hematoxylin and eosin and examined microscopically. Indirect immunohistochemical staining for IL15 (R&D Systems), NOS2 (Cayman Chemical, Ann Arbor, MI, USA), IL6 and CD163 (both Novus Biologicals, Litleton, CO, USA), were performed as previously described (Roberts et al., 2003 (link); Marcondes et al., 2007 (link)). Colorimetric development was performed with the NovaRed chromagen (Vector) followed by a hematoxylin counterstain (Sigma-Aldrich).

The localization of GPER in the initial segment (IS), caput, corpus and cauda tissues was investigated by immunohistochemistry using a protocol similar to one previously described [3 (link)]. Tissue was paraffin embedded and sectioned at a thickness of 5 μm. Antigen retrieval was performed by submerging slides in a citric acid based antigen unmasking solution (Vector Laboratories, Inc., Burlingame CA) in Coplin jars and steam heated to 93 °C for 5 min after which they were allowed to cool to room temperature. Endogenous peroxidase activity was blocked by incubation in 0.3 % hydrogen peroxide in methanol for 30 minutes. After a blocking step, tissues were incubated for two hours at room temperature with rabbit anti-human GPR30 (1:2500; sc-48525; Santa Cruz Biotechnology). Following primary antibody incubation, sections were incubated with goat anti-rabbit biotinylated secondary antibody followed by an avidin–biotin horseradish peroxidase complex (Vector Laboratories). Immunostaining was visualized using NovaRed chromagen (Vector Laboratories). Pre-absorbed primary antibodies were substituted for primary antibody as a negative control; all other steps were identical including exposure times with NovaRed. All slides were counterstained with Immunomaster Hematoxylin and evaluated by light microscopy.

Paraffin-embedded mouse liver tissue cut to 3 μm thickness was stained with H&E using standard protocols. For immunohistochemical studies, paraffin-embedded mouse liver sections were incubated with optimally diluted rabbit antimouse vimentin, rabbit antimouse desmin, rabbit antimouse F4/80 (all Abcam, Cambridge, UK) or rat antimouse CD45 (eBioscience, Hatfield, UK) using a Dako Autostainer. Staining was detected with immPRESS antirabbit secondary antibody and visualised in NovaRED chromagen (both Vector Laboratories, Peterborough, UK). Sections were then counterstained with haematoxylin (VWR International, Leighton Buzzard, UK). For CD45+ immune cell quantification, five non-overlapping fields from each stained section were captured using a light microscope (Zeiss, Germany) with identical illumination and exposure. Digital image analysis was performed using ImageJ software. CD45 values were expressed as the percentage of the total area of the section occupied by CD45 staining.