Mouse anti glutamine synthetase

Eyes were fixed in 4% paraformaldehyde for 5 minutes and then anterior segments were removed to form an eyecup. After post fixation in 4% paraformaldehyde for 1 hour, eyecups were transferred to PBS containing 30% sucrose and then embedded in optimal cutting temperature (OCT) compound. Immunohistochemistry was performed on 10-μm thick cryosections, as described previously.²⁰ Antibodies used were mouse anti-glutamine synthetase (1:500; Abcam, Cambridge, UK); rabbit anti-light ferritin (E17) (1:200; a gift from P. Arosio, University of Brescia, Italy); goat anti-albumin (1:100; Betyl Laboratories, Montgomery, TX, USA); rat anti-transferrin receptor (1:200; Serotec, Kidlington, UK); and rat anti–glial fibrillary acidic protein (anti-GFAP) (1:500; Abcam). Control sections were treated identically but with omission of primary antibody. Sections were analyzed by fluorescence microscopy using identical exposure parameters across genotype using Nikon Elements software (Nikon Instruments, Melville, NY, USA). Pixel density analysis of the l-ferritin (Ft-L) stain was completed using an open source image-processing package (FIJI software).^{24 (link)}

Protein concentration in hippocampal extracts from mice and human tissue was
measured using the BCA Protein Assay Kit (Cole-Parmer). Forty micrograms of
protein/lane were electrophoretically separated on a 12% SDS polyacrylamide gel
and electrically transferred onto a Hybond-P PVDF transfer membrane (Millipore)
for 1 h. Membranes were blocked in PBS-milk 5% at RT for 1 h. Next, membranes
were incubated in block solution overnight with the following primary
antibodies: mouse anti-glutamine synthetase (1:500; Abcam), mouse anti-GAPDH
(1:1,000; Abcam), rabbit anti-Cyclophilin B (1:1,000; Sigma) and rabbit
anti-β-actin (1:1,000; Abcam). Membranes were incubated for 1 h with IRDye 680CW
goat anti-mouse antibody, IRDye 800CW goat anti-mouse antibody, or IRDye 800CW
goat anti-rabbit antibody (LI-COR, 1:20,000), then scanned with an Odyssey
infrared imaging system (LI-COR) and analyzed using Un-Scan-It gel version 6.1
(Silk Scientific).