Rbpms

The following reagents were used: Streptozotocin (Merck, Darmstadt, Germany); AdipoRon (Selleckchem, Houston, TX, USA); a triglyceride assay kit and a non-esterified free fatty acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China); and dihydroethidium (Thermofisher, MA, USA). The following antibodies were used: Rabbit monoclonal to AdipoR1 (EPR6626,Abcam, Cambridge, UK); rabbit polyclonal to RNA-binding protein with multiple splicing (RBPMS; Abcam); rabbit monoclonal to glutamine synthetase (EPR13022(B),Abcam); chicken polyclonal to glial fibrillary acidic protein (GFAP; Abcam); rabbit polyclonal to EGR4 (Abcam); phospho-AMPKα rabbit monoclonal antibody (mAb) and AMPKα rabbit mAb (40H9/D5A2,Cell signaling, Boston, USA); phospho-acetyl CoA carboxylase (ACC) rabbit mAb and ACC rabbit mAb (D7D11/C83B10,Cell signaling); cleaved caspase-3 rabbit mAb (Cell signaling), β-actin (8H10D10,Cell signaling); donkey anti-rabbit AlexaFluor-594 and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch, Philadelphia, USA). The following reagents were used: 5,6-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Thermofisher); RNAimax (Thermofisher); Opti-MEM I (Thermofisher); small interfering RNA (siRNA; GenePharma, Shanghai, China); Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan); and 4ʹ,6-diamidino-2-phenylindole (DAPI; Absin, Shanghai, China).

The protocol for immunohistochemistry as previously described.⁴⁷ The retinas were incubated in PBS with 1% Triton X-100 and 0.5% Tween 20 for 1 h at room temperature and in 4% BSA for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: RBPMS (1:500, Abcam), ChAT (1:100, Abcam), VGlut2 (1:100, Frontier Institute), syntaxin (1:100, Abcam), tyrosine hydroxylase (1:100, Abcam) and Prox1 (1:100, AngioBio) in blocking buffer. Secondary anti-rabbit, mouse, and goat IgG, conjugated with Alexa TM488, TM594, and 633, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat mounted, and the sections were mounted on slide glass. The TUNEL assay was performed based on our previous reports.⁴⁸ (link)^,49

Retinas were dissected, flat-mounted onto nitrocellulose membranes (Membrane Filter Black, white grid; GE Healthcare Life Sciences, Whatman TM, Chicago, IL, USA) with the ganglion cell layer upwards and fixed in 4% PFA. Immunolabelling for retinal ganglion cells (RGCs) was achieved using an antibody against RNA-binding protein with multiple splicing (RBPMS, 1:500, Abcam, Cambridge, UK; Cat #ab194213; RRID:AB_2920590).
To determine RGC cell densities, images were taken from all four retinal quadrants at three different locations (central, middle and peripheral positions corresponding to 1/6, 1/2 and 5/6 of the retinal radius), in order to adjust for local variance in RGC densities. Quantification was then performed manually from the images in an observer-blinded manner using an Image J cell counting plug-in (https://imagej.nih.gov/ij/).