Mouse antibody against cry1

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.

Hypothalamic tissue samples (n = 3/group) were fixed in 10% buffered formalin and embedded in paraffin wax. Sections (4 μm) were then cut and then dewaxed and dehydrated using a graded series of ethanol concentrations. Sections were then covered in 25 mM citrate buffer (pH 6.0), boiled and then allowed to cool for 40 min. Hydrogen peroxide (3%) was used to inactivate endogenous peroxidase activity. Sections were then blocked and permeabilized with normal goat serum (Abcam, Cambridge, MA, USA) for 30 min at room temperature. Next, 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). The next day, sections were incubated with an HRP-conjugated anti-mouse or anti-rabbit secondary antibody (Boster Biological Technology, Wuhan, China) for 30 min. Color was developed with a solution of 3–3-diaminobenzidine (Dako Denmark A/S, Glostrup, Denmark), and the results were assessed with a Dako REAL EnVision Detection System. The specificity of antibody binding was evaluated using negative controls (incubation with PBS without antibody). Finally, areas showing positive antibody binding were quantified using ImageJ software v3.91 (NIH, Bethesda, MD, USA).