Fitc conjugated goat anti mouse igg

ARPE-19 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium: nutrient mixture F12 (hereafter named DMEM:F12), fetal bovine serum (FBS), bovine serum albumin (BSA), trypsin-EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate, gentamycin, phosphate-buffered saline (PBS), penicillin, streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), Tween-20 and PAP pen were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-labeled annexin V (to bind PS), annexin V-binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂, were purchased from Becton Dickinson Biosciences (BD), Belgium. Minimum essential medium (MEM) was purchased from Invitrogen (Carlsbad, CA, USA). Pipettes, 25 cm² flasks, 15 mL and 50 mL centrifugation tubes, 1 L glass bottles, and pipette tips were supplied by VWR International (West Chester, PA, USA). Vacuum filtration rapid filter mix was supplied by BioNordika (Oslo, Norway). Mouse anti-RPE65, rabbit anti-occludin, FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG antibodies were obtained from Abcam (Cambridge, UK). Mouse anti-ZO-1 and Alexa Fluor 568 phalloidin were purchased from Life Technologies (Carlsbad, CA, USA).

Slides were fixed with Histochoice (Amresco). The DeadEnd^™ Fluorometric TUNEL System (Promega, Madison, WI) was used for TUNEL assay according to the manufacturer’s instruction. Nuclei of cells were counterstained with DAPI (Vector Labs). For MVD assay, slides were labeled overnight with primary antibodies. Fluorescent IHC was performed first using monoclonal mouse antihuman anti-CD31 antibody (1:200, Dako), anti-EGFR antibody (1:250, Abcam), anti-BrdU antibody (1:250, Abcam), anti-Ki67 antibody (1:200, Abcam) or isotype control antibodies followed by FITC conjugated goat-anti-mouse IgG (1:1000, Abcam) or Cy5 conjugated goat-anti-mouse IgG (1:1000, Abcam) according to manufacturer’s protocols. The images were analyzed on the BioView Duet fluorescent scanning station using oil objective and DAPI/FITC/Rhodamine single band filters (Semrock). For each piece of the slide, the experiment was performed for three independently times.

Cells were washed three times with 0.01 M phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min. After the removal of excess paraformaldehyde, cells were blocked and permeabilized for 1 h using a mixture of 0.1% Triton X-100 (Fisher Scientific) and 10% goat serum (Sigma) in PBS. The cells were separately incubated overnight at 4°C with different primary antibodies; the primary antibodies for neuron, astrocyte, and BMEC detection were anti-microtubule associated protein 2 (MAP2, Abcam, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP, Abcam), and anti-von Willebrand factor (VWf, Abcam), respectively. The tight junction protein was labeled with anti-zonula occludens-1 (ZO-1, Proteintech, Chicago, IL, USA) antibody overnight at 4°C. The following day, cells were incubated with secondary antibodies for 1 h (Alexa Fluor 647-conjugated goat anti-chicken IgY, Abcam; FITC-conjugated goat anti-mouse IgG, Abcam; Alexa Fluor 555-conjugated donkey anti-rabbit IgG, Abcam; and Alexa Fluor 488-conjugated goat anti-rabbit, Proteintech). Cells nuclei were stained with 4′,6-diamidino-2-phenylindole (Solarbio, Beijing, China) at a concentration of 0.5 μg/mL for 10 min. Fluorescence images were captured with a fluorescence microscope (FluoView 1000, Olympus, Tokyo, Japan).

The cells were fixed and permeabilized following the same procedure as cytoskeleton staining. Then, they were blocked with 1% BSA in PBS for 1 h and incubated with rabbit polyclonal primary antibodies against zo-1, claudin-1, cadherin-E, and cadherin-N (1 : 100 Abcam, England) and a mouse polyclonal primary antibody against vimentin (Abcam, England) overnight at 4°C. Secondary antibodies were used, including TRITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG (1 : 200 Abcam, England). They were incubated for 2 h at 37°C in a humidified atmosphere in the dark. The nuclei of the TM cells were stained with DAPI (1 : 1000; Sigma, USA). The samples were analyzed in a drop of PBS under a fluorescence microscope at this state.

An indirect IFA was performed to confirm expression of constructs in RTG-2 cells.. RTG-2 cells were cultured in six-well plates until cell density was 60%–80% confluent. Cells were transfected with 2 μg of plasmids per well using Lipofectamine® 2000 Transfection Reagent (Invitrogen, USA). Cells that were transfected with pcDNA3.1 empty vector served as negative controls. At 48 h after transfection, cells were fixed with 2% paraformaldehyde for 12 min and washed with PBS buffer. Subsequently, cells were incubated with corresponding polyclonal antibodies at 1:100 dilutions at 37 °C for 1 h. The control group was incubated with pool polyclonal antibodies. After three washes with PBS, cells were incubated with FITC-conjugated Goat Anti-Mouse IgG (Abcam, USA) at 37 °C for 1 h. After four washes with PBS, cells were observed under fluorescent microscope.