Donkey anti mouse alexa 647

Sections containing the NAc (Bregma 1.70 to 0.90 mm) or the amygdala (Bregma -1.40 to -2.00 mm) were incubated overnight at 4°C with primary antibodies diluted in the blocking solution: rabbit anti-GFP (Abcam #290, 1:5000) and mouse anti-TH (Millipore #MAB318, 1:500) and, after three washes in the blocking solution, incubated with secondary antibodies diluted in the blocking solution overnight at 4°C: donkey anti-rabbit-Alexa 488 and donkey anti-mouse-Alexa 647 (Thermofisher Scientific, 1:500). Sections were mounted in Prolong Gold antifade mountant (Thermofisher Scientific). For synaptic α4-YFP puncta detection, sections were incubated overnight at room temperature with mouse anti-synaptophysin antibody (Sigma #S5768, 1:200), followed by 4h incubation with goat anti-mouse monovalent Fab-Alexa 647 (Jackson #115607003, 1:500). Free mouse epitopes were blocked with anti-mouse unconjugated monovalent Fab antibodies (Abcam #ab6668, 1:100) overnight at 4°C. GFP and TH immunolabelling were conducted as above with goat-raised secondary antibodies (Thermofisher Scientific, 1:500).

Protein samples were resolved on 4% to 20% Mini-Protean TGX precast protein SDS-PAGE gels (Bio-Rad Laboratories; Hercules, CA) and blotted on polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were incubated in TBS buffer containing 0.1% (vol/vol) Tween 20 (TTBS) and 5% (wt/vol) blocking reagent (Bio-Rad) with gentle shaking at room temperature for 30 min. Primary antibodies were added as described above with mouse anti-strep (Millipore Sigma no. 71590-M, 1:1,000 dilution) and mouse anti-His antibodies (Santa Cruz Biotechnology, Dallas, TX; no. SC-53073, 1:400 dilution) diluted in TTBS with 0.5% (wt/vol) blocking reagent, followed by incubation with gentle shaking at room temperature for 12 to 14 h. Membranes were washed three times with TTBS, followed by incubation (2 h at room temperature with gentle shaking) with secondary antibodies (goat anti-rabbit-HRP [Thermo Fisher Scientific no. 65-6120, diluted 1:3,000] and donkey anti-mouse-Alexa 647 [Thermo Fisher Scientific no. A-31571, diluted 1:1,000]) diluted in TTBS with 0.5% (vol/vol) blocking reagent. Membranes were washed twice in TTBS and once in TBS before Western blot detection. Blots were visualized using the Bio-Rad ChemiDoc MP imaging system.

Transduced SCN slice cultures were fixated with a prechilled 4% paraformaldehyde (PFA) solution and washed with PBS. For immunostaining, tissue samples were incubated with a DeepLabel antibody staining kit (Logos Biosystems, South Korea) permeabilization solution for 6 h and then labeled overnight with primary antibodies: rabbit-anti-PER2 (1:500, Merck Millipore), mouse-anti-NeuN (1:500, Merck Millipore), and DAPI (1:10,000, Thermo Scientific) at 37 °C with agitation. After washing with PBS three times, the appropriate secondary antibodies, donkey anti-rabbit-Alexa-594 and donkey anti-mouse-Alexa-647 (Invitrogen), were applied for 6 h at room temperature. The slice tissues were then imaged using a confocal LSM700 microscope (Carl Zeiss, Germany).

Eyes were enucleated and a small incision (4 mm) was made posterior to the limbus and the eye was fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 30 min. Subsequently, an incision was made along the entire circumference of the limbus to remove the entire anterior segment. The optic cups were fixed in 4% PFA for an additional 3 hours. After fixation, the tissue was rinsed with 70% ethanol and later embedded in paraffin. Seven-micron sagittal retinal sections through the optic nerve head were obtained, de-paraffinized in xylene, and rehydrated with a graded series of ethanol. Following permeabilization with 0.1% Triton X-100 and blocking with 5% normal donkey serum containing 5% BSA in PBS, retinal sections were incubated with the appropriate primary antibodies overnight. Primary antibodies used were rabbit anti-Cox 17 (1:100, #ab69611, Abcam), mouse anti-ATP5H (1:200, #ab110275, Abcam)and goat anti-Brn3a (1:200, #sc31984, Santa Cruz Biotechnology). Secondary antibody incubation for 1 hr was carried out at room temperature. Secondary antibodies used were either donkey anti-rabbit Alexa 546 (1:1000, Invitrogen), donkey anti-mouse Alexa 647 (1:1000, Invitrogen) or donkey anti-goat 488 (1:1000, Invitrogen). Retinal sections incubated with no primary antibody served as the blank to assess non-specific staining by the secondary antibodies.