Ab150185

Immunohistochemistry (IHC) was performed as previously reported [51 (link)]. For immunofluorescence in cultured astrocytes, cells were fixed with 4% paraformaldehyde (15 min, 0°C), rinsed with glycine solution (30 mM in 0.1 M PBS solution), and permeabilized with Triton X-100 (0.2%, 3 min). Preincubation was performed with 10% NGS for 1 h at 37°C. The following primary antibodies were used: mouse anti-NeuN (1:1000; Abcam #ab104224), mouse anti-GFAP (1:500, Millipore #MAB360), rabbit anti-GFAP (1:500, Proteintech #16825-1-AP), guinea pig anti-S100β (1:500, Synaptic Systems #287 004), rabbit anti-Ezrin (1:100, Cell Signaling #3145), mouse anti-vGluT1 (1:200, Millipore #MAB5502), and rabbit anti-PSD95 (1:100, Invitrogen #51–6900). The following Alexa conjugated secondary antibodies were used: goat anti-mouse 488 (1:1,000, Abcam #ab150113), goat anti-rabbit-488 (1:1,000, Abcam #ab150077), goat anti-rabbit 647 (1:500, Abcam #ab150079), and goat anti-guinea pig 488 (1:500, Abcam #ab150185). Immunofluorescence of Ezrin was performed by using an Alexa Fluor 488 Tyramide SuperBoost Kit (Invitrogen #B40912 and #B40941) according to the manufacturer’s protocols. After nucleus labeling with DAPI, the coverslips were mounted on slides using anti-fade solution. For quantification of fluorescent intensity, sections from the two groups were stained and imaged with exactly the same protocol.

At different time points post intravitreal injection, the mice were killed and perfused with saline and 4% PFA. Eyes were then enucleated and fixed in 4% PFA for 2 h at room temperature. Subsequently, retinas were isolated from the eyecups, and each retina was divided into four quadrants. Next, retinas were incubated with blocking buffer containing 5% bovine serum albumin (BSA) and 0.5% Triton-X 100 in PBS (PBS-T) for 90 min at room temperature. Then, retinas were incubated overnight at 4 ℃ with guinea pig anti-RBPMS (#1832-RBPMS,1:100, PhosphoSolutions, Denver, CO, USA) in blocking buffer. After being fixed in 4% PFA for 10 min and washed three times in PBS-T, the retinas were incubated with the secondary antibody (goat anti-guinea pig Alexa Fluor 488, ab150185, 1:1000, Abcam, Cambridge, UK) in blocking solution for 90 min at room temperature. Finally, retinas were washed in PBS three times and mounted with the mounting medium containing DAPI. Then, retinal whole-mounts were examined with a fluorescence microscope (Leica, Wetzler, Germany). RBPMS-positive RGCs were quantified by two observers using ImageJ software in three areas (center, middle, periphery) in each of the four quadrants of the retina.

Kidney sections were incubated at room temperature with FLEX polyclonal guinea pig anti-insulin antibody (Agilent, IR002) for 1 h at room temperature. The sections were gently washed five times using the blocking buffer and then incubated with recombinant rabbit anti-glucagon antibody (Abcam, ab92517) in blocking buffer (1:570) for 1 h. The sections were then washed five times gently with blocking buffer and incubated with goat anti-guinea pig immunoglobulin G (IgG) (H+L) secondary antibody (Alexa Fluor 488) (Abcam, ab150185; 1:250) and goat anti-rabbit IgG H&L (Alexa Fluor 647) (Abcam, ab150079) in blocking buffer (1:250) for 1 h at room temperature. After gently washing the sections with 4 °C DPBS with Ca²⁺ and Mg²⁺, the sections were labeled with 4′,6-diamidino-2-phenylindole (Millipore Sigma, F6057), coverslipped (Chase Scientific, ZA0294) and placed at 4 °C for 2 h before imaging.