Vectashield plus antifade mounting medium

S2 cells were seeded on cell culture dishes with glass bottom and transfected with indicated plasmids. The cells were washed twice with 1×PBS, fixed with 3.7% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 15 min. Thereafter, the cells were washed with 1×PBS and incubated with blocking buffer (1% bovine serum albumin and 0.1% Tween 20) for 30 min. Subsequently, the cells were incubated with primary antibodies at 4 °C overnight. After washing with 0.1% Tween 20 in PBS 4 times, the cells were incubated for 1 h with secondary antibody. Primary antibodies used are as follows: rabbit anti-PER (1:1,000, a gift from Dr. Joanna Chiu) and mouse anti-HA (1:1,000, Medical & Biological Laboratories). Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit (1:1,000, Abcam) and Alexa Fluor 594 goat anti-mouse (1:1,000, Abcam). Nuclei were counterstained with Hoechst 33342 (Beyotime). Finally, the cells were rinsed with 0.1% Tween 20 in PBS 4 times and mounted with Vectashield Plus Antifade Mounting Medium (Vector Laboratories). The samples were scanned by the Olympus FV3000 confocal microscope with a 100× silicone oil objective.

Whole eyes were excised, and corneas were punctured, followed by incubation in 4% paraformaldehyde (PFA, pH 7.5) for 30 min. Eyes were washed with PBS and incubated at 4 °C in 30% sucrose solution containing 0.05% sodium azide. Eyes were embedded in optimal cutting temperature compound, flash frozen, and sectioned. Cryosections (10 μm) were fixed in 2% PFA, permeabilized in PBS with 0.1% Triton-X-100. To visualize NF-κB nuclear localization, MIO-M1 cells were cultured on chamber slides (CELLTREAT) for 24 h prior to exposure to hyperglycemia conditions. Cells were then fixed in 4% PFA and permeabilized with PBS with 0.1% Triton-X-100. Sections or cell monolayers were then blocked with 10% normal donkey serum and labeled with the appropriate antibodies (Table S1). Slides were mounted with Vectashield Plus Antifade Mounting Medium with DAPI mounting media (Vector labs). Images were captured using an SP8 confocal laser microscope (Leica Microsystems) with frame-stack sequential scanning. ImageJ (National Institutes of Health) was used to estimate macrophage counts from three separates fields of view per retina. Thresholds were set to isolate CD80- or F4/80-positive cells, and particles were counted. Single-stained population counts were also confirmed manually.

Cells seeded on coverslips were fixed with 4% paraformaldehyde for 20 min followed by washing with PBS, permeabilized with PBS containing 0.5% Triton-X 100, and blocked in 3% BSA for 1 h at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies for pMED1 (1:500), pAR (1:100), and methyl C (1:50). Coverslips were washed three times with PBS-T and incubated with the respective Alexafluor secondary antibodies (1:200) for 1 h at room temperature followed by counterstaining with DAPI (Sigma). Coverslips were mounted on glass slides using VECTASHIELD PLUS Antifade Mounting Medium (Vector Laboratories) and cured overnight. Images were captured on a Zeiss LSM 880 confocal microscope. FIJI software was used to integrate the signal for quantitative image analysis. For specific quantification of nuclear staining, regions of interest were overlaid with the DAPI signal, and only co-localizing regions were included in the integration to exclude background cytoplasmic signals.

Mice were deeply anesthetized with isoflurane (2%) and transcardially perfused with phosphate-buffered saline (PBS), followed by 10% neutral buffered formalin (NBF). The animals were decapitated, and the whole heads were incubated overnight in 10% NBF at 4 °C before the fiber optic was removed and the brain was extracted from the skull. Brains were incubated in 10% NBF for up to 4 h followed by 30% sucrose (w/v in PBS) for cryoprotection. 50 µm coronal brain sections were obtained using a cryostat (Leica Biosystems, ON, CA, Cat. #CM1850 UV). The sliced brain sections were collected in a staggered fashion and placed into 3 consecutive wells. Sections were rinsed before and between incubations with 0.1 M PBS. Sections were incubated in blocking solution (5% donkey serum in PBS with 0.5% Triton-X 100 (PBST), Sigma-Aldrich, St. Louis, MO, USA) for 1 h and blocking solution (5% donkey serum in 0.3% PBST) was used in subsequent antibody incubations. Controls were included where the primary antibody was omitted to check for non-specific binding of the secondary antibodies. Free-floating brain sections were then mounted onto Superfrost^TM slides in VECTASHIELD® PLUS Antifade Mounting Medium (Vector Laboratories Inc., Newark, CA, USA, Cat. #VECTH190010) and cover-slipped. All primary and secondary antibodies, along with their conjugates, are presented in Supplementary Table 1.

To address the presence or not of the APLi.GAL4-driven UAS-TNT and UAS-mCherry products in the APL silencing experiment (Figure 4), whole flies were fixed on formaldehyde (FA) 4% in PBS with 0.1% Triton X-100 (PBT 0.1%) immediately after in vivo imaging. Once all animals sustained the in vivo imaging protocol, brains were dissected using a pair of forceps in a small Petri dish covered with a layer of silicon, fixed for further 20 min on FA 4% in PBT 0.1%, washed 3 times for 5 min ca in PBT 0.1% and mounted with Vectashield Plus Antifade Mounting Medium (Vectorlabs) on 76 × 26 mm microscope slides (Thermo Scientific) with 1# coverslips (Carl Roth). Brains were oriented with the dorsal part facing upwards. Imaging was performed on an LSM 780 confocal microscope (Zeiss) equipped with a Plan-Apochromat 63×/1.4 Oil DIC M27 objective (Zeiss). 512 × 512 pixels images were acquired, covering a region of the brain big enough to include the APL soma and branches around the MB calyx and MB lobes. Brains and their related in vivo imaging data were assigned to the classes ‘APL OFF’ or ‘APL ON’ based on the presence or not of the TNT-co-expressed mCherry fluorescence, respectively.