For histological analysis, fixed lymph nodes were dehydrated in 100% ethanol and embedded in paraffin (Leica) using a Leica EG1150 tissue embedder. 7-μm-thick sections using a HM340E Thermo Scientific microtome were processed for hematoxylin-eosin (HE) (Leica) and immunohistochemistry staining as described (41 (link)). For immunohistochemistry of histology slides the following antibodies were used: anti-TER-119 (R&D Systems, MAB1125) and Alexa Fluor 488 conjugated goat anti-rat IgG antibody (Life Technologies A11006). Stained slides were mounted with Vectashield DAPI Mounting Medium (Vector Laboratories, H-1200). Tissue section samples were imaged using a Nikon Ni-U upright microscope (Nikon Instruments) using a 40x dry objective connected to a Nikon DS-Ri2 camera.
Mab1125
Manufactured by R&D Systems
Sourced in United States
MAB1125 is a monoclonal antibody that recognizes human CD80 (B7-1). CD80 is a costimulatory molecule expressed on antigen-presenting cells that provides a signal for T cell activation.
Automatically generated - may contain errors
Lab products found in correlation
Af3628, by R&D Systems (2 mentions)
Alexa fluor 555 conjugated 70 kda dextran, by Thermo Fisher Scientific (2 mentions)
P0799, by Merck Group (2 mentions)
Dylight fluor coupled secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Ds ri2, by Nikon (1 mentions)
Eg1150 tissue embedder, by Leica camera (1 mentions)
Alexa fluor 488 conjugated goat anti rat igg antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield dapi mounting medium, by Vector Laboratories (1 mentions)
Hm340e, by Thermo Fisher Scientific (1 mentions)
Ab16645, by Abcam (1 mentions)
Af1935, by R&D Systems (1 mentions)
Nb100 417, by Novus Biologicals (1 mentions)
Isolectin gs ib4 alexa fluor 568 conjugate, by Thermo Fisher Scientific (1 mentions)
Ab9610, by Merck Group (1 mentions)
Polyclonal rabbit anti zo 1, by Thermo Fisher Scientific (1 mentions)
Mab1320, by R&D Systems (1 mentions)
Mouse monoclonal anti claudin 5, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Ab119341, by Abcam (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
7 protocols using mab1125
1
Histological Analysis of Lymph Nodes
2
Vascular Network Visualization in Embryos
3
Comprehensive Immunohistochemical Profiling
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For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R&D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R&D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB). The secondary antibodies that were used were donkey anti-mouse Alexa488 (715-545-150, 1:400, Jackson ImmunoResearch), goat anti-mouse Alexa488 (115-545-146, 1:400, Jackson ImmunoResearch), goat anti-rat Alexa568 (A11077, 1:400, Invitrogen), donkey anti-rabbit Alexa647 (711-605-152, 1:400, Jackson ImmunoResearch). Blood vessels were visualized using Isolectin GS-IB4 Alexa Fluor 568 conjugate (I21412, 1:250, Invitrogen). Images were collected on a confocal microscope (Zeiss LSM 510 unit mounted on an Axiovert 200 M inverted microscope) with 10x/0,3 EC Plan-NEOFLUAR Objective and/or with 20x/0,8 Plan-APOCHROMAT and on a Nikon AR1 confocal microscope with 40x/1,3 Plan-Fluor Objective. Image processing was performed using Zen 2011 and the NIH ImageJ software.
4
Retinal Vascular Analysis in OIR Models
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For flat-mounted retinas, eyes were enucleated from mice and fixed in 4% PFA for 1 h at 4 °C. Retinas were dissected, washed with PBS and permeabilized with PBS containing 1% TritonX-100 overnight at 4°C then blocked in PBS containing 2% BSA, 0.3% TritonX-100 for 12 h at 4 °C. After blocking, for visualization of retinal vasculature in OIR models, flat-mounted retinas were stained with isolectinGS-IB4 (1:100) for 2 h at RT. For IF staining of red blood cells, blood vessels, macrophage and microglia, flat-mounted retinas were incubated in blocking solution with following primary antibodies at 4 °C overnight: rat anti-TER119 monoclonal (1:200, MAB1125, RD, US); rat anti-CD105 monoclonal (1:200, MAB1320, RD, US); rabbit anti-F4/80 monoclonal (1:200, 30325S, Cell Signaling Technology, US); mouse anti-Claudin-5 monoclonal (1:200, 35-2500, Invitrogen, US) and rabbit anti-ZO-1 polyclonal (1:300, 61-7300, Invitrogen, US). After washing, the retinas were incubated with corresponding secondary antibodies (1:300, Jackson ImmunoResearch, US) for 2 h at RT. Flat-mounted retinas were analyzed using a confocal fluorescence microscope (LSM 800, Carl Zeiss, Germany).
5
Vascular Perfusion Analysis in Embryos
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E12.5 mutant and control embryos underwent cardiac perfusion with Alexa Fluor 555-conjugated 70 kDa dextran (50 μL of 2.5 mg/mL in PBS; Invitrogen) using a pulled glass pipette with mouth connector and tubing (Sigma, P0799) in the course of the 15 to 20 minutes when a visible heart beat could be observed. Fluorescent visualization of the entire vasculature confirmed the perfusion. Tracer was left circulating for 10 minutes at room temperature, embryos were decapitated, their heads fixed in 4% paraformaldehyde overnight at 4°C, cryosectioned and stained as described above. Staining was performed with the following primary antibodies; CD31/PECAM1 (R&D systems, AF3628) 1:1000 and monoclonal TER-119 (R&D Systems, MAB1125) 1:250 followed by a 2 hours incubation at room temperature with DyLight Fluor-coupled secondary antibodies (1:1000, Thermo Scientific Pierce).
6
Conditional Exoc3l2 Knockout in Retinal Vasculature
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VE-cad-CreER mice [16 (link)] were mated with floxed Exoc3l2 mice to obtain conditional Exoc3l2 KO mice. Forty μg 4-hydroxytamoxifen was subcutaneously injected at postnatal day 2, 3.5 and 5. Littermates were used as controls.
Whole-mounted retinas were prepared according to a previous report [17 (link)]. Briefly, enucleated eyes were fixed for 20 min in 4% paraformaldehyde at room temperature. A small hole was made in the cornea using a 27-gauge needle, and a circular incision was made using fine scissors. Then, retinal cups were dissected from the eyes and postfixed for 30 min in 4% paraformaldehyde at 4 °C. The primary antibodies used were rabbit anti-GFP (Alexa 488-conjugated; Molecular Probes), biotinylated isolectin B4 (1:500; B-1205; Vector Laboratories), mouse anti-Ter119 (1:250; MAB1125; R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-neuron-glial antigen-2 (NG2) (1:200; AB5320; Millipore-Sigma, Burlington, MA, USA) and hamster anti-Pecam1 (1:1000; ab119341; Abcam, Cambridge, UK). The secondary antibodies were suitable species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin coupled to Alexa Fluor dyes (Invitrogen, Waltham, MA, USA).
Whole-mounted retinas were prepared according to a previous report [17 (link)]. Briefly, enucleated eyes were fixed for 20 min in 4% paraformaldehyde at room temperature. A small hole was made in the cornea using a 27-gauge needle, and a circular incision was made using fine scissors. Then, retinal cups were dissected from the eyes and postfixed for 30 min in 4% paraformaldehyde at 4 °C. The primary antibodies used were rabbit anti-GFP (Alexa 488-conjugated; Molecular Probes), biotinylated isolectin B4 (1:500; B-1205; Vector Laboratories), mouse anti-Ter119 (1:250; MAB1125; R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-neuron-glial antigen-2 (NG2) (1:200; AB5320; Millipore-Sigma, Burlington, MA, USA) and hamster anti-Pecam1 (1:1000; ab119341; Abcam, Cambridge, UK). The secondary antibodies were suitable species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin coupled to Alexa Fluor dyes (Invitrogen, Waltham, MA, USA).
7
Cryosectioning and Immunostaining Protocol
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Preparing for cryosection, eyes were fixed with 4% paraformaldehyde (PFA) at room temperature for 4 h. Specimens were embedded in OCT compound (Sakura, Tokyo, Japan) and sectioned at 8 μm. For retinal whole-mount, eyes were fixed with 4% PFA at room temperature for 1 h. After dissection, retinas were transferred to cold methanol for further use. Preparing for cellular staining, endothelial cells seeded on glass coverslips were fixed in 4% PFA at room temperature for 15 min. Following fixation, samples were permeabilized with PBS containing 0.2% Triton X-100 for 30 min, blocked with PBS containing 3% bovine serum albumin for 1 h, and incubated with primary antibodies at 4 °C for 12–48 h. Primary antibodies used for immunofluorescence analysis were as follows: anti-TBK1 (Abcam, ab40676), FITC-isolectin B4 (IB4) (MilliporeSigma, L2895), anti-CD31 (Santa Cruz Biotechnology, sc376764), anti-collagen type IV (ab6586), anti-TER119 (R&D SYSTEMS, MAB1125), anti-VE-Cadherin (R&D SYSTEMS, AF1002; MAB9381), and anti-ZO-1 (Invitrogen, 33-9100). After wash out with PBS, samples were incubated with secondary antibodies at room temperature for 2 h. Images were captured using an inverted confocal microscope (Olympus FV3000).
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