Mouse anti psa ncam

Human retinas were dissected from donor eye bulbs and fixed with 4% formaldehyde for 4 h at room temperature. After overnight incubation in 30% sucrose in 1× PBS, retinas were embedded in optimal cutting temperature (OCT) compound (Hartenstein, Würzburg, Germany) and sliced into 12‐μm‐thick sections. For staining, slides were dried at room temperature for 10–15 min and washed two times in PBS. Slices were blocked with 10% bovine serum albumin (BSA; Sigma), 5% goat serum (Invitrogen), and 0.1% Triton X‐100 for 20–30 min. Slices were incubated in one of the following primary antibodies overnight at 4°C: rabbit anti‐Iba1 (1:1,000; Wako), mouse anti‐PSA‐NCAM (1:500, polysialic acid; Millipore), rat anti‐PSA (CLONE 12F8, 1:200; BD Pharmingen), or mouse anti‐oligosialic acid (CLONE 105, 1:200; Invitrogen), and anti‐SIGLEC11 (1:500, clone 3EH, binding up to a dilution of 1:128K to the SIGLEC11‐specific peptide ISISHDNTSALE) (Shahraz et al, 2015). Slices were washed with PBS three times and then incubated with the corresponding Cy3‐conjugated secondary antibody (Jackson) for 4 h at room temperature. After three washing steps in PBS, slices were incubated in DAPI for 15 min at room temperature and mounted with Moviol.

Primary antibodies were used as follows: goat anti-DCX (1 : 100; Santa Cruz, Dallas, TX, USA), mouse anti-MAP2 (1 : 100; Millipore, Billerica, MA, USA), rabbit anti-Iba1 (1 : 2000; Wako, Richmond, VA, USA), mouse anti-6E10 (1 : 1000, Covance, San Diego, CA, USA), mouse anti-phospho tau (1 : 1000, Pierce, Rockford, IL, USA), goat anti-tau (C17) (1 : 1000, Santa Cruz), rat anti-neprilysin (1 : 500, R&D Systems, Inc.), mouse anti PSA-NCAM (1 : 2000, Millipore), rabbit anti-VEGF (1 : 1000, Santa Cruz), mouse anti-PSD-95 (1 : 2000, Thermo Scientific, Waltham, MA, USA), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 10000, Ab Frontier, Seoul, South Korea).

Slides were incubated with primary antibodies diluted in blocking solution overnight at 4 °C, rinsed, and incubated with the secondary antibodies for 1 h at room temperature. Primary antibodies were the following: rabbit anti-CD31 (1:100, BD Biosciences), rabbit anti- VEGFR2 (1:200, Abcam), rabbit anti-DCX (1:200, Cell Signaling), mouse anti-GFAP (1:200, Abcam), mouse anti-PSA-NCAM (1:200, Millipore), mouse anti-α smooth muscle actin (1:500, Sigma), mouse anti-acetylated α tubulin (1:1000, Sigma), rabbit anti-β catenin (1:500 Abcam), rabbit anti-phosphorylated EGFR (1:200, Cell Signaling), and rabbit anti-Ki67 (1:200, Abcam). For nuclear staining 4′,6-diamidino-2-phenylindole (DAPI) 500 ng/ml (Sigma) was used. Secondary antibodies were Alexa Fluor dye-conjugated goat anti-mouse or goat anti-rabbit IgG or donkey anti-goat IgG (diluted 1:200 in blocking solution, Jackson ImmunoResearch or Invitrogen). Confocal images were taken on LSM 510 META NLO (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). The following filter setting is applied: FITC Ch2 band pass (BP) 500–550 IR Texas Red Ch3: long pass (LP) 595 DAPI Ch2: BP 435–485 IR. For low magnification fluorescence micrographs Nikon E800 and 80i upright microscopes with Spot RT cooled CCD cameras were used.