Eyes were enucleated, and retinae were dissected and fixed in 4% formaldehyde for 30 min at room temperature. Fixed retinae were cryoprotected in sucrose in PBS for a few hours and embedded in optical cutting temperature compound on dry ice. Sections (20 μm thick) were cut on a cryostat (Leica). Retinal sections or whole retinal cups were blocked in 5% BSA in PBST (PBS with 0.1% Triton X-100), stained with primary antibodies at 4 °C overnight, and washed three times with PBST. Primary antibodies used in this study included rabbit anti-red/green opsin (1:300, AB5405; EMD Millipore); goat anti-blue opsin (1:100, sc-14365; Santa Cruz Biotechnology Inc.); rabbit anti-GFAP (1:500, Z0344; DAKO); rabbit anti-Iba1 (1:1,000, PA5-21274; ThermoFisher), and rhodamine-conjugated and FITC-conjugated PNA (1:1,000; Vector Laboratories). Sections were stained using secondary antibodies, including donkey anti- rabbit CY3, donkey anti-rabbit Alexa Fluor 647, and donkey anti-goat Alexa Fluor 647 (all used at 1:1,000; Jackson ImmunoResearch), and were costained with DAPI in the dark for 2 h at room temperature and mounted in Fluoromount-G (SouthernBiotech). Images were taken using a 40× objective with Z-stacks on a Zeiss LSM780 confocal microscope. Images used for comparison between groups were taken side by side at the same confocal settings.
Donkey anti goat alexa fluor 647
Manufactured by Jackson ImmunoResearch
Sourced in United States
Donkey anti-goat Alexa Fluor 647 is a secondary antibody conjugated with the fluorescent dye Alexa Fluor 647. It is designed to detect and visualize the presence of goat primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti rabbit, by Jackson ImmunoResearch (3 mentions)
Donkey anti rabbit alexa fluor 647, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 647 donkey anti mouse, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Cryostat, by Leica (1 mentions)
Ab5405, by Merck Group (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Sc 14365, by Santa Cruz Biotechnology (1 mentions)
Z0344, by Agilent Technologies (1 mentions)
Rabbit anti iba1, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Volocity version 6, by PerkinElmer (1 mentions)
Lsm 5 duo, by Zeiss (1 mentions)
8 protocols using donkey anti goat alexa fluor 647
1
Immunohistochemical Analysis of Retinal Cell Types
2
Visualizing T Cell Migration Across Endothelium
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CFSE-stained T cells (5 × 104 cells per transwell) migrated for 4 h across endothelial monolayers to CCL19 (0.5 μg ml−1), fixed for 20 min at 4 °C with 4% (w/v) paraformaldehyde (Affymetrix), and paraformaldehyde then neutralized for 10 min at 4 °C with PBS 0.1 M glycine (Sigma-Aldrich), pH 7.4. Cells were permeablized with PBS 0.2% (v/v) Triton X-100 (Sigma-Aldrich), stained for 30 min at 4 °C with Alexa fluor 555-phalloidin (Life Technologies) and washed with PBS containing 0.2% (v/v) Triton X-100 followed by PBS alone. In some experiments cell layers were also stained with polyclonal goat anti-VCAM-1 at 2 μg ml−1 (sc-1504, Santa Cruz) and detected with Donkey anti-goat Alexa Fluor 647 at 5 μg ml−1 (Jackson Immunoresearch, 705-606-147). Transwell membranes were transferred onto glass slides and visualized by confocal microscopy (Zeiss LSM 510 Meta, and LSM5 Duo) with a × 63 or × 40 objective, respectively. Z-stack images were acquired every 1 μm with 2-μm slice thicknesses for examination of in vitro endothelial monolayers. Images were analysed with Volocity version 6.1.1 software (Perkin Elmer).
3
Quantifying Connexin43 Phosphorylation
4
Optimized Antibody Staining for Tissue Optical Clearing
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The following antibodies used in tissue optical clearing experiments were diluted in 0.5% bovine serum albumin (BSA), 10% dimethyl sulfoxide (DMSO), and 0.5% Triton-X-100 in PBS: rabbit anti-SARS-CoV N (Rockland Immunochemicals, #200–401-A50, 1:500), mouse anti-SARS-CoV NP (Sino Biological, #40143-MM05, 1:400), rat anti-I-A/I-E (Biolegend, #107601, 1:400), mouse anti-CD68 (Invitrogen, #MA5-13324, 1:400), rat anti-CD68 (BioLegend, #137001, 1:400), rat anti-Ki-67 (Biolegend, #652401, 1:200), rabbit anti-vWF (Dako, #A0082, 1:1,000), rabbit anti-Cleaved Caspase-3 (Cell Signaling, #9661, 1:200), and rabbit anti-Uteroglobin (Abcam, #ab40873, 1:500). Isotype control rabbit (Biolegend, #910801), rat (Biolegend, # 400602), and mouse (Biolegend, #401402) antibodies were used. Secondary antibodies were diluted in 2% donkey serum, 10% DMSO, and 0.5% Triton-X-100 in PBS at 1:1,000. All the following secondary antibodies were purchased from Invitrogen unless stated otherwise. Donkey anti-rabbit Alexa Fluor 568 (#A10042), donkey anti-mouse Alexa Fluor 568 (#A10037), donkey anti-rabbit Alexa Fluor 647 (#A31573), donkey anti-rat Alexa Fluor 647 (Jackson ImmunoResearch, #712–605-153), donkey anti-goat Alexa Fluor 647 (#A21447), donkey anti-rabbit Alexa Fluor 790 (#A11374), donkey anti-rat Alexa Fluor 790 (Jackson ImmunoResearch, #712–655-153).
5
Fluorescent Antibody Labeling Protocols
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Alexa Fluor–coupled antibodies (Thermo Fisher Scientific and Molecular Probes): Alexa Fluor 488 donkey anti–mouse (A21202), Alexa Fluor 488 donkey anti–rabbit (A21206), Alexa Fluor 488 donkey anti–goat (A11055), Alexa Fluor 568 donkey anti–mouse (A10037), Alexa Fluor 568 donkey anti–rabbit (A10042), Alexa Fluor 568 donkey anti–goat (A11057), Alexa Fluor 647 donkey anti–mouse (A31571), Alexa Fluor 647 donkey anti–rabbit (A31573), Alexa Fluor 647 donkey anti–goat (A21447); Jackson ImmunoResearch Laboratories, Inc., antibodies: aminomethylcoumarin donkey anti–chicken (703-155-155) and rhodamine red-X donkey anti–rat (712-295-153). For LI-COR Biosciences Odyssey WBs, Jackson ImmunoResearch Laboratories, Inc., donkey anti–mouse (680; 715-625-151), donkey anti–rabbit (790; 711-655-152), donkey anti–sheep (680; 713-625-147), donkey anti–rat (680; 712-625-153), and donkey anti–goat (800; 926-32214; LI-COR Biosciences) antibodies were used.
6
ccRCC FFPE Immunofluorescence Protocol
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5-micron thickness cut ccRCC Formalin-Fixed Paraffin-Embedded (FFPE) sections were deparaffinized and rehydrated using xylene, high to low percentages of ethanol, and finally placed in 1× PBS. The heat antigen retrieval method was applied using 1 mM EDTA for at least 25 min. 5% Donkey serum and 1% BSA was used as blocking buffer and as primary and secondary antibodies diluent. Antibodies for CA9 Rabbit (#NB100-417; Polyclonal; Novus Bio) at 1:350, CA9 Goat (#PA5-47268, Polyclonal; Invitrogen) at 1:50, VIM Chicken (#NB300-223; Polyclonal; Novus) at 1:150, WNT5a/b Rabbit (#55184-1-AP, Polyclonal; Proteintech) at 1:100, CP Goat (#A80-124A; Polyclonal; Bethyl lab) at 1:100, and PCSK6 Rabbit (#PA5-32966; Polyclonal; Invitrogen) at 1:100 were applied on sections and later detected with specific fluorescent secondary antibodies conjugated with Alexa Fluor 488 Donkey anti-Rabbit #711-546-152, Alexa Fluor 488 Donkey anti-Chicken #703-606-155, Alexa Fluor 488 Donkey anti-Goat #705-546-147, Alexa Fluor 594 Donkey anti-Rabbit #711-586-152, Alexa Fluor 594 Donkey anti-Goat #705-586-147, Alexa Fluor 647 Donkey anti-Goat #705-607-003 from Jackson ImmunoResearch diluted at 1:1000. All IF images were taken using a Lecia DMi8 fluorescence microscope.
7
Immunohistochemistry of Drosophila Larval Tissues
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Immunohistochemistry was performed as previously described (Matthews et al., 2007 (link)). Third instar larvae were dissected in 1X PBS, fixed in 4% paraformaldehyde (Electron Microscopy Sciences; CAS #30525-89-4) in PBS for 15 minutes, rinsed three times in PBS-TX (0.3% Triton X-100 in PBS), and blocked for 1 hour at 4°C in normal donkey serum (1:20; Jackson Immunoresearch; RRID: AB_2337258). Primary antibodies used were chicken anti-GFP (1:1000; Abcam; RRID: AB_300798), rabbit anti-DsRed (1:500; Takara Bio, RRID:AB_10013483), goat anti-HRP (1:200; Jackson Immunoresearch; RRID: AB_2338952), mouse anti-Coracle (1:10; Developmental Studies Hybridoma Bank; RRID:AB_1161642, RRID:AB_1161644) and mouse anti-Elav (1:10; Developmental Studies Hybridoma Bank; RRID:AB_528217). Secondary antibodies used were Alexa Fluor 488 donkey anti-chicken (1:200; Jackson Immunoresearch; RRID: AB_2340375), Rhodamine Red-X donkey anti-rabbit (1:200; Jackson Immunoresearch; RRID: AB_2340613), Alexa Fluor 647 donkey anti-goat (1:200; Jackson Immunoresearch; RRID: AB_2340437), Rhodamine Red-X donkey anti-goat (1:200; Jackson Immunoresearch; RRID: AB_2340423), Rhodamine Red-X donkey anti-mouse (1:200; Jackson Immunoresearch; RRID: AB_ AB_2340831), and Alexa Fluor 647 donkey anti-mouse (1:200; Jackson Immunoresearch; RRID: AB_2340862).
8
Antibody Panel for VEGFR2, NRP1, and CD34 Analysis
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Anti‐VEGFR2 antibody (AF357; R&D Systems, Minneapolis, MI, USA) was used at a 1:100 dilution in an in situ proximity ligation assay (PLA). Anti‐VEGFR2 (2479; Cell Signaling Technology, Danvers, MA, USA) was used for immunofluorescent staining (IF) at a 1:150 dilution. Anti‐NRP1 antibody (60067–1; Proteintech, Rosemont, IL, USA) was used for PLA at a 1:100 dilution. Anti‐NRP1 (AF566; R&D Systems) at a 1:100 dilution was used for IF. Dylight‐650 conjugated anti‐CD34 (NBP2‐44567C; Novus Biologicals, Littleton, CO, USA) was used for in situ PLA at a 1:100 dilution. Anti‐CD34, clone QBEnd10 (IR63261‐2; Agilent Technologies, Santa Clara, CA, USA) was used for IF at a 1:100 dilution. Secondary antibodies, Alexa Fluor 488 donkey anti‐mouse, Alexa Fluor 647 donkey anti‐mouse, Alexa Fluor 555 donkey anti‐rabbit, Alexa Fluor 488 donkey anti‐goat (Invitrogen, Carlsbad, CA, USA), and Alexa Fluor 647 donkey anti‐goat (Jackson ImmunoResearch, Philadelphia, PA, USA) were used for IF at a 1:400 dilution, (see supplementary material, Table S1 for details). Isotype control antibodies used for IF were the following: goat IgG (0500–000‐003, Jackson ImmunoResearch), rabbit IgG (2729, Cell Signaling Technology), and mouse IgG1, kappa (554 121, BD Biosciences, San Jose, CA, USA).
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