Alexa fluor 594 conjugated donkey anti rabbit antibody

At 6 h and 1 day post-CNV induction, enucleated mouse eyes were fixed in 4% paraformaldehyde for 1 h at room temperature, cryoprotected in 30% sucrose at 4°C overnight, and embedded in OCT compound. Eyes were cryosectioned to 15 μm thickness. For immunofluorescence, sections were blocked in a blocking buffer (0.5% Triton, 0.2% BSA, and 5% donkey serum in PBS) for 1 h at room temperature and subsequently incubated with primary antibodies overnight at 4°C. After washing, samples were incubated with secondary antibodies for 1 h at room temperature. Goat anti-Iba1 antibody (1:250; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, 011-27991), rabbit anti-TMEM119 (1:500, Synaptic Systems, Göttingen, Germany, 400 002), rat anti-CD31 (1:100, BD Biosciences, NJ, USA, 553370), and rabbit anti-MMP-9 antibody (1:100; Abcam, Cambridge, MA, USA, ab38898) were used for primary antibodies, and Alexa Fluor 488-conjugated donkey anti-goat antibody (1:500; Thermo Fisher Scientific, Waltham, MA, USA, A11055), Alexa Fluor 594-conjugated donkey anti-rabbit antibody (1:500; Thermo Fisher Scientific, A21207), and Alexa Fluor 647-conjugated donkey anti-rat antibody (1:500; Jackson Immuno Research Laboratories, INC., PA, USA, 712-605-15) were used for secondary antibodies.

All the fluorenylmethyloxycarbonyl (Fmoc) amino acids and Fmoc-Lys (Boc)-Wang Resin were purchased from AAPPTec (Louisville, KY, USA). Dichloromethane (DCM), acetic acid, acetic anhydride, thioanisole, phenol, hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA), N-trityl-1,2-ethanediamine, phenol, thioanisol, dimethylformamide (DMF), N,N′-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), iodine, methyl tert-butyl ether (MTBE), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), and dexamethasone (DEX) were purchased from Sigma-Aldrich (St Louis, MO). Hematoxylin and eosin (H&E) stains were purchased from MilliporeSigma (St Louis, MO). Rabbit anti-pANXA2 (phospho-Tyr24) antibody was purchased from Signalway Antibody (College Park, MD). AlexaFluor 594-conjugated donkey anti-rabbit antibody was purchased from Thermo Fisher Scientific (Waltham, MA).