Alexa fluor 594 goat anti rabbit igg

HCVECs or mouse choroidal flats plated on gelatin‐coated glass slides were fixed with 4% paraformaldehyde (PFA), permeabilized with 2% bovine serum albumin (BSA; A7030, Sigma Aldrich) and 0.5% Triton X‐100 in PBS, blocked with background buster (NB306, Innovex Biosciences, USA) and incubated overnight at 4°C with primary antibodies anti‐AR, anti‐PEDF, anti‐LR and anti‐α‐SMA, followed by secondary antibodies Alexa Fluor 594 goat‐anti‐rabbit IgG (111‐585‐144, Jackson ImmunoResearch Inc., USA), Alexa Fluor 488 goat‐anti‐mouse IgG (115‐545‐003, Jackson ImmunoResearch Inc.), along with DAPI (C1005, Beyotime, China) staining. Digital images have been acquired utilizing a fluorescence microscope (Leica microsystem, Germany). Co‐localization of AR/DAPI or PEDF/LR was analysed using the included Leica LAS‐AF software.

Mice were anesthetized with an intraperitoneal injection of 250 mg/kg dose of freshly prepared Tribromoethanol (Avertin). They were then transcardially perfused with ice-cold phosphate-buffered saline (PBS) to wash out the blood, and following the discoloration of liver, the buffer was replaced to freshly prepared ice-cold 4% paraformaldehyde (PFA) with the assistance of circulating pump to supply sufficient perfusion pressure. After 20 min perfusion, the brain tissue was extracted from the skull and was fixed overnight in 4% PFA. The fixed brain tissue was placed in 30% sucrose for 48 hr at 4°C. Brain tissue was then submerged into OTC compound (Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. 30 µm coronal sections were cut using a cryotome (Leica) and mounted on Fisherbrand Superfrost Plus Microscope Slides. Sections were permeabilized using 0.25% Triton-x100 in PBS for 30 min, washed three times in PBS, and incubated with primary antibody against hepcidin overnight at 4°C. Sections were then washed three times in PBS and incubated with Alexa Fluor594 Goat Anti-Rabbit IgG at 1:200 (Jackson ImmunoResearch) for 2 hr at room temperature. Nuclei were counterstained with DAPI containing ProLong Gold Antifade mounting media (Invitrogen) and images of frontal cortex were acquired using a Zeiss Axio Observer.Z1 fluorescence microscope.