Provis ax70

Paraformaldehyde (4% PFA, Merck) fixed cells were permeabilized with ice-cold methanol for 10 min. After a 30-min block with 4% bovine serum albumin (BSA), cells were incubated for 60 min at room temperature (RT) with primary antibodies (see Table 1), i.e., anti-myelin basic protein (MBP) (1:250, Serotec, Oxford, United Kingdom) or anti-TG2 (1:500, Ab2, Labvision, Fremont, CA, United States) diluted in 4% BSA in PBS. Next, the cells were rinsed with PBS and incubated for 25 min with appropriate TRITC-conjugated secondary antibody (1:50, diluted in 4% BSA in PBS; Jackson ImmunoResearch, Westgrove, PA, United States). Nuclei were stained with DAPI (1 μg/ml, Sigma-Aldrich, United States), and mounting medium (Dako, Heverlee, Belgium) was added to prevent image fading. The cells were analyzed with a conventional fluorescence microscope (Olympus ProVis AX70 or Leica DMI 6000 B). OLGs were characterized by morphology, i.e., cells with a typical astrocytic morphology were excluded, and in each experiment at least 250 cells were scored as either MBP-positive or MBP-negative (“differentiation”). In addition, positive cells bearing MBP-positive membranous structures spread between the cellular processes were identified as myelin membrane-forming, irrespective of the extent of membrane formation (“myelination”).

Immunostaining protocol was adapted from a published protocol [19 (link)]. Mouse eyes were enucleated and fixed in 4% PFA at room temperature for 15 minutes, then transferred to PBS for 10 minutes. After whole eye fixation, retinas were dissected and fixed with cold methanol (-20°C) for 20 minutes, followed by blocking in Perm/Block solution (PBS + 0.3% Triton + 0.5% BSA + 5% goat serum). Goat anti-mouse PECAM-1 (1:50) (R&D Systems, Fischer Scientific, #AF3628) combined with Alexa Fluor 594-conjugated isolectin GS B4 (1:200) (Invitrogen, #I21413) in Perm/Block solution was used for primary antibody incubation, followed by Alexa Fluor 488-conjugated donkey anti-goat antibody (1:1000) (Invitrogen, #A32814) for secondary antibody incubation. After extensive washing, retinas were mounted using ProLong Glass Antifade (Invitrogen) onto a coverslip and allowed to set overnight. Immunofluorescence was captured via Olympus Provis AX-70 and Leica TCS SP8 Confocal microscopes. The fluorescence intensity was quantified using ImageJ software.