Dmrxa2

Immunohistochemical staining was performed as previously described [36 (link)]. The scaffold without and with cells were washed in PBS, fixed in 4% paraformaldehyde, decalcified dehydrated and embedded in paraffin. Sections were cut at a thickness of 5 ​μm and were stained with H&E after deparaffination. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 20 ​minutes at room temperature. Antigen retrieval was then performed with citrate buffer at 80°C for 10 ​minutes. Primary antibodies against GFP (1:100; Santa Cruz, CA, USA) and osteocalcin (1:100; Santa Cruz, CA, USA) were used. Donkey anti-goat IgG horseradish peroxidase (HRP)-conjugated secondary antibody and goat anti mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:200) was then added for an hour, followed by 3, 3’ diaminobenzidine tetrahydrochloride (DAKO, Glostrup, Denmark) in the presence of H₂O₂ for signal detection. Afterward, the sections were rinsed, counterstained in hematoxylin, dehydrated with graded ethanol and xylene, and mounted with p-xylene-bis-pyridinium bromide (DPX) permount (Sigma Aldrich, St Louis, MO, USA). Primary antibody was replaced with blocking solution in the negative controls. All incubation times and conditions were strictly controlled. The sections were examined under light microscopy (DMRXA2, Leica Microsystems Wetzlar GmbH, Germany).

Immunostainings were analyzed using a microscope equipped with appropriate filter settings for detecting Cy5, Alexa 555, and DAPI. Fluorescence images were obtained via a nonconfocal microscope (DMRXA2, Leica Microsystems) equipped with Metamorph v 2.1.39 (Olympus). Light microscopy images were grabbed with a Leica DC200 camera using Leica DC200 software (Leica). The software program ImageJ was used to fusion the images, and the image editing software Microsoft publisher was used to combine the obtained images into panels. The reference of the various brain structures was made according to the Franklin and Paxinos atlas “Mouse brain in stereotaxic coordinates” (39 ).
For the quantification of c-Fos immunoreactivity to light, the number of c-Fos-positive neurons in the SCN was counted by two independent experimenters blind to experimental conditions in an area of 300 × 500 µm at levels corresponding to bregma −0.30 to −0.70 mm (rostral to caudal part of the SCN) (Fig. 2 A, viii). Approximately nine brain sections from each animal containing the SCN (with AVP costaining) were analyzed, and c-Fos–positive cells were visually counted using a grid on each image. C-Fos immunoreactivity in response to light was analyzed using two-way ANOVA (light pulse and genotype conditions) followed by post hoc t tests.