Rat anti cd31

HUVECs were grown on a cover slide until confluent and then fixed with 4% paraformaldehyde. Tumor cryosections or the fixed HUVECS were blocked and permeabilized in PBS containing 1% bovine serum albumin (BSA) and 0.3% TritonX-100, then immunostained with rat anti-CD31 (1:400, BD, USA) antibody at 4°C for overnight. After washing with PBS, sections were incubated with FITC-conjugated goat anti-rat secondary antibody(1:500, Abcam, USA) at room temperature for 2 hours, followed by Hoechst staining. Images were captured with a fluorescence microscope.

Images were acquired using an Olympus FLUOVIEW FV1000 confocal microscope. Fluorescence intensity was measured using ImageJ or Olympus FLUOVIEW software. The Pearson’s coefficient for co-localization was measured using Olympus FLUOVIEW software. Commercially available antibodies used for immunoblotting and immunostaining of tissue sections include rabbit anti-MFSD2a (Cell Signaling Technology, Beverly, MA), mouse anti-E-cadherin (cat#610181, BD, Biosciences, San Diego, CA, USA), rabbit anti-NG2 (Millipore), rabbit anti-GFAP (cat#Z0334, DAKO, Glostrup, Denmark), chicken anti-Nestin (cat#CH23001, Neuromics, Minneapolis, MN, USA), rat anti-CD31 (cat#553370, BD Biosciences, San Diego, CA, USA), rat anti-CD34 (GTX28158, GeneTex, Irvine, CA, USA), mouse-anti-Erk1/2 (cat#9107 S, Cell Signaling Technology), and rabbit-anti-pErk1/2 (cat#4370 s, Cell Signaling Technology). Secondary antibodies include anti-rabbit IgG Alexa Fluor-594/488, 1:500 (cat#711-585-152/cat#711-545-152, Jackson labs), anti-rat IgG Alexa Fluor-594, 1:500 (cat#112-585-167, Jackson labs), anti-mouse IgG Dylight-405, 1:500 (Jackson labs), and anti-rabbit IgG DyLight-405,1:500 (cat#711-475-152, Jackson labs). Secondary antibodies used for immunoblotting were IRDye 800CW anti-rabbit, 1:10,000 (LICOR) and IRDye 680RD anti-mouse, 1:10,000 (LICOR).

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.

The choroid plexus was explanted 48 h post adoptive cell transfer from the fourth ventricle and transferred on glass object slides and incubated in PBS (PAN Biotech) + tween20 (0.3%; Sigma) at RT for 5 min, washed twice in PBS (PAN Biotech) for 5 min and fixed in PBS (PAN Biotech) + PFA (2,2%; Sigma), glucose (2%, Sigma), sodium acide (0.02%; Sigma) for 20 min at RT. Choroid plexus were then rinsed in PBS (PAN Biotech), fixed in methanol (100%, Sigma) for 6 min at RT, washed twice in PBS for 5 min and blocked in PBS (PAN Biotech) + BSA (1%; PAN Biotech), tween20 (0.3%; Sigma), normal goat serum (10%; Sigma) for 30 min at RT. The staining was performed in PBS (PAN Biotech) + tween20 (0.3%; Sigma) + primary antibody for 2h at RT (rat anti CD31; 1:100; BD). After washing the tissue twice in PBS (PAN Biotech) for 5 min, secondary stainings were performed using anti rat secondary antibodies (donkey anti rat-Alexa647; life technologies). Finally, the tissue was stained with DAPI (1 µg/ml; Sigma) and embedded in fluorescent mounting media (Dako).