Expression of TLR4 in the rat pineal cryosections was performed accordingly to da Silveira Cruz-Machado et al.22. Briefly, animals were deeply anesthetized by intramuscular injection of ketamine (160 mg/kg) plus xylazine (40 mg/kg) and perfused transcardially with 150 mL saline solution followed by 1000 mL of cold 4%paraformaldehyde fixative solution, pH 9.5. The perfusion was performed at two time-points at LED Canopy Light phase (ZT2 and ZT10) or three different time points at dark phase under red light (ZT14, ZT18, ZT22). It is noteworthy to mention that in this experiment we have waited 2 hours after lights off because of differences between coding gene expression to protein translation. Each pineal gland was removed from the skull and cryoprotected in the same fixative solution plus 20% sucrose overnight, followed by 30% sucrose in PBS for 14 hr, embedded in Tissue Tek freezing medium, rapidly frozen in dry ice and stored at -80 °C. Cryostat sections (20 µm) were fixed for 30 min in freshly prepared 4%paraformaldehyde in PBS. Following 5 min of incubation with 0.1 M glycine, sections were incubated with blocking solution (3% albumin and 0.01% saponin in PBS) for 1 hr at room temperature. The Avidin-Biotin Blocking kit (Vector, SP2001, Burlingame, CA, USA) was used to block endogenous biotin. Rabbit polyclonal antibody against rat TLR4 (1:200; Abcam) was incubated overnight at 4 °C followed by incubation with appropriated secondary antibody conjugated to biotin (1:200; Abcam) for 1 hr at room temperature. The Avidin-Biotin-Complex (Vectastain Elite kit; Vector, PK-6105, Burlingame, CA, USA) staining system was used to localize biotinylated antibody. Peroxidase activity was revealed with 3, 3′-diaminobenzidine (DAB substrate kit for Peroxidase; Vector, SK-4100) according to manufacturer instruction. The sections were counterstained with methylene blue. Controls were performed by the omission of the primary antibodies from the procedure. Staining was completely abolished under these conditions.

　　Data analysis

　　Data are presented as mean ± S.E.M. Statistical analysis was performed using the unpaired Student’s t test or ANOVA followed by Newman–Keuls post-test. Values of p < 0.05 were considered statistically significant.

　　Additional Information

　　Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.