Cy3 conjugated goat anti rabbit igg secondary antibody

For immunofluorescence staining, sections (4 μm) of hypothalamic tissue (n = 3/group) were dewaxed and hydrated using a graded series of ethanol concentrations, boiled in 25 mM citrate buffer (pH 6.0) for 10 min and then cooled with cold deionized water for 1 h. Sections on coverslips were then washed three times in PBS and blocked and permeabilized with normal goat serum (Abcam, Cambridge, MA, USA) for 30 min at room temperature. After drying, sections were incubated overnight at 4°C with rabbit antibodies against BMAL1 and CLOCK (1:100; Abcam, Cambridge, MA, USA) and a mouse antibody against CRY1 (1:100; Santa Cruz Biotechnology, CA, USA). All antibodies were used at a dilution of 1:100. The next day, sections were washed three times in PBS and then incubated with Cy3-conjugated goat anti-rabbit IgG secondary antibody and Cy3-conjugated goat anti-mouse IgG secondary antibody (Boster Biological Technology, Wuhan, China) for 1 h at 25°C. Coverslips were then washed three times with PBS, mounted on glass slides with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime; Shanghai; China) and visualized using a BX53 microscope (Olympus, Tokyo, Japan). Areas of positive antibody binding were then quantified using ImageJ software v3.91 (NIH, Bethesda, MD, USA). A schematic diagram of the experimental process is shown in Figure 1.

IL-17, Bax and caspase-3 expression levels were detected in paraffin-embedded RV sections using the anti-IL17 antibody (Abcam, UK, 1:20 dilution), anti-Bax antibody (Proteintech, Wuhan, China, 1:100 dilution) and anti-caspase-3 antibody (Bioss, Beijing, China, 1:200 dilution), protein expression was visualized using Alex Fluor 488 Goat Anti-Rabbit secondary antibodies (R&D, CA, USA, 1:200 dilution, green) or Cy3 Conjugated Goat Anti-Rabbit IgG secondary antibody (BOSTER biological technology, Wuhan, China, 1:100 dilution, red). Nuclei were counterstained with DAPI.

Lentivirus (Biovector, China), Src-shRNA (GenePhama, China), Cy3 Conjugated Rabbit Anti-Goat IgG Secondary Antibody (Boster, China), 98% tanshinone IIA (Sigma-Aldrich, USA), Src antibody (#2109) (Cell Signaling, Inc., USA), p-Src antibody (#2101) (Cell Signaling, Inc., USA), p-ERK1/2 antibody (#4695) (Cell Signaling, Inc., USA), and p-AKt antibody (#4059) (Cell Signaling, Inc., USA) were used. BLAB/C-nu/nu nude mice purchased from China Vital River, Inc. (Beijing, China), were used in this study.