Led light source

Cells (1 × 10⁴/well) were seeded in 96-well black glass bottom plates (Cellvis, Mountain View, CA, USA) and left untreated or treated with TNF-α (10 ng/mL, 15 min) at a 70–80% confluency point. Cells were fixed with 2% PFA for 10 min, permeabilized and blocked with 2% BSA in PBS-1X consisting of 0.1% triton X-100 for 1 h at room temperature. APC-conjugated NF-kB (14G10A21 Biovision, Milpitas, CA, USA), 1:400 diluted in blocking buffer) was incubated at 4 °C in humidified chamber overnight. Next day, they were washed twice with PBS-1x and subsequently incubated with NucBlue Live stain reagent for 1h in the dark. Pictures were taken with an inverted Nikon TI2 microscope (Melville, NY, USA), LED light source ( Lumencor, Parkway Beaverton, OR, USA), and CMOS camera (prime 95B, Photometrics, Tucson, AZ, USA), and analyzed using Fiji imageJ software.

Whole eyes were isolated from euthanized mice and fixed overnight in 4% paraformaldehyde. The tissue was dehydrated, cryopreserved in sucrose and embedded in optimal cutting temperature (OCT) compound. A Shandon Cryotome E cryostat was used to prepare 15-micron sections through the optic nerve head. Sections were permeabilized with 0.5% Triton X-100/PBS and then incubated overnight at 4 °C with primary rabbit antibodies against glial fibrillary acidic protein (GFAP; Agilent Technologies (Santa Clara, CA, USA), catalog #Z033429-2) at 1:500 dilution in 10% donkey serum/0.5% Triton X-100/PBS. Sections were then rinsed in PBS and incubated with secondary antibodies (conjugated to Alexa Fluor 594; Thermo Fisher Scientific (Waltham, MA, USA)) under the same conditions. Sections were then mounted in a medium containing DAPI (Dako Fluorescence Mounting Medium, Agilent Technologies (Santa Clara, CA, USA)). Single field light microscopy images were acquired with a Nikon Eclipse Ti inverted microscope (Nixon, Minato City, Tokyo, Japan) coupled to an LED light source (Lumencor, Beaverton, OR, USA) using a 40× (N.A. 1.4) objective and processed with the NIS element software package. Relative fluorescence from acquired images was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and were processed using Adobe Illustrator (Adobe Inc., San Jose, CA, USA).