After dewaxing in xylene and rehydration in an alcohol series, the sections were boiled in citrate buffer (pH 6) in a pressure cooker for 3 minutes for antigen retrieval, followed by incubation in 0.6% H2O2 to inactivate endogenous peroxidase activity. For BrdU detection, the tissue was additionally pre-treated with 2 N HCl followed by borate buffer (pH 8.5). Sections were then incubated with primary antibodies in blocking buffer (TBS containing 0.1% Triton X-100 and 3% donkey serum) either overnight at +4 °C or for 1 hour at room temperature. The antibodies used were as follows: anti-mouse BrdU (1:500 dilution; DAKO); anti-rabbit Ki-67 (1:150; Merck Millipore); goat anti-CD31 (1:150; R&D Systems, Minneapolis, MN, USA), and anti-rabbit Iba1 (1:2000; Wako Industries). This was followed by the addition of the appropriate biotinylated secondary antibody (1:250; Vector Laboratories, Burlingame, CA, USA). After incubation with avidin-biotin solution (Vectastain ABC Elite kit; Vector Laboratories), the antigen was visualised by developing in DAB (Saveen Werner AB, Malmö, Sweden). The sections were then dehydrated in an alcohol series, cleared in xylene, and cover-slipped in Xtra-kitt mounting medium (Medite GmbH, Burgdorf, Germany). The human biopsy samples were treated in a similar way.
Goat anti cd31
Manufactured by R&D Systems
Sourced in United States
Goat anti-CD31 is a primary antibody that binds to the CD31 antigen, also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule). CD31 is a transmembrane glycoprotein expressed on the surface of endothelial cells, platelets, monocytes, and some T cell subsets. This antibody can be used for the identification and quantification of cells expressing CD31 in various applications such as flow cytometry, immunohistochemistry, and Western blotting.
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Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Rabbit anti ve cadherin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti occludin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica camera (1 mentions)
Ab7260, by Abcam (1 mentions)
Af3628, by R&D Systems (1 mentions)
Goat anti doublecortin, by Santa Cruz Biotechnology (1 mentions)
Gb11138, by Wuhan Servicebio Technology (1 mentions)
Fish skin gelatin, by Merck Group (1 mentions)
Anti mouse igg 488, by Thermo Fisher Scientific (1 mentions)
Dapi counterstain, by Southern Biotech (1 mentions)
Donkey anti goat 488, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Stereo investigator software, by MicroBrightField (1 mentions)
Cryoembedding medium, by Thermo Fisher Scientific (1 mentions)
Anti rat alexafluor488, by Thermo Fisher Scientific (1 mentions)
10 protocols using goat anti cd31
1
Immunohistochemical Staining Protocol
2
Immunofluorescence Staining of Heart Sections
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The heart sections were incubated in blocking solution for 1 h at room temperature and then incubated at 4 °C overnight with one of the following antibodies: rabbit anti-VE-cadherin (1:200; Invitrogen Cat. #36-1900), goat anti-CD31 (1:200; R&D Cat. #AF3628), rabbit anti-zonula occludens-1 (1:200; Invitrogen Cat. #61-7300), rabbit anti-occludin (1:200; Invitrogen Cat. #71-1500), mouse anti-NG2 (1:100; Millipore Cat. #AB5320), rabbit anti-VCAM-1 (1:200; Santa Cruz Cat. #sc-13160), rabbit anti-MPO (1:50; Abcam Cat. #ab9535), mouse anti-CD68 (1:100; Abcam Cat. #ab955), rabbit anti-iNOS (1:200; Abcam Cat. #ab15323), rabbit anti-CD206 (1:100; Abcam Cat. #ab64693), or mouse anti-cTnT (1:200; Abcam Cat. #ab8295). Confocal images were captured at room temperature with ZEN software on an upright confocal microscope (LSM 700; Carl Zeiss) using the predefined ZEN software configurations for Alexa Fluor 546, Alexa Fluor 488, and DAPI.
3
Immunofluorescent Labeling of Brain Sections
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Brain sections were incubated overnight at 4° in the following primary antibodies: rabbit polyclonal anti-PDGFA (1:50; Bioss Antibodies), sheep polyclonal anti-PRKAR1B (1:50; R&D Systems), goat anti-COL-IV (1:50; EMD Millipore), and goat anti-CD31 (1:50; R&D Systems). Sections were immersed in deionized water for 3 min at 37° and then treated with 0.5 mg/ml pepsin in 0.2N HCl for 15 min at 37°. Slides were then washed twice in 1× PBS for 10 min at room temperature. With the exception of anti-COL-IV, antibodies were diluted in 0.5% PBTB (1× PBS, 0.0.5% Triton X-100, and 0.5% BSA) containing 10% normal donkey serum. Anti-COL-IV was diluted in 0.5% PBS/Tween 20 (PBT). Sections were washed three times in 0.5% PBT and then incubated for 2 hr at room temperature with their respective secondary antibodies (donkey anti-rabbit Alexa Fluor 594, donkey anti-goat Alexa Fluor 488, and donkey anti-sheep Alexa Fluor 594 in a 1:1000 dilution; Life Technologies). All sections were then counterstained with DAPI (1:1000 in 1× PBS) and washed with 1× PBS prior to mounting with Aqua PolyMount. Images were taken using a Leica SP5 confocal microscope located within the imaging facility at The Jackson Laboratory.
4
Mapping Cellular Tropism of AAV-PHP.eB
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To determine what kinds of cells in the brain AAV-PHP.eB could transduce, sections were stained with rabbit anti-NeuN (GB11138, Servicebio, Wuhan, China), rabbit anti-GFAP (ab7260, abcam, Cambridge, MA, USA), goat anti-Doublecortin (Santa Cruz Biotechnology, Dallas, Texas, USA), and goat anti-CD31 (AF3628, R&D Systems, Minneapolis, MN, USA).
The preparation of sections for immunofluorescent staining was as follows: sections were 1) rinsed in PBS at RT for 5 min; 2) blocked in 5% bovine serum albumin (BSA) in PBS at RT for 1 h; 3) incubated with primary Abs (diluted in 5% BSA) at RT overnight, including the rabbit anti-NeuN Ab (1:100), rabbit anti-Glial fibrillary acidic protein (GFAP) Ab (1:100), goat anti-Doublecortin (DCX) Ab (1:100), and goat anti-CD31 Ab (1:100); 4) washed in PBS, 10 min × 3 times; and, 5) incubated in secondary Abs (diluted in 5% BSA) at RT for 1 h. For primary Abs from rabbit, Alexa Fluor 488-labeled goat anti-rabbit IgG (1:100) were used. Alexa Fluor 488-labeled Donkey anti-goat IgG (1:100) was used for goat primary Ab; 6) washed in PBS, 10 min × 3 times; and, 7) coverslipped using anti-fade mounting medium with or without 10 µg/ml Hoechst dye.
The preparation of sections for immunofluorescent staining was as follows: sections were 1) rinsed in PBS at RT for 5 min; 2) blocked in 5% bovine serum albumin (BSA) in PBS at RT for 1 h; 3) incubated with primary Abs (diluted in 5% BSA) at RT overnight, including the rabbit anti-NeuN Ab (1:100), rabbit anti-Glial fibrillary acidic protein (GFAP) Ab (1:100), goat anti-Doublecortin (DCX) Ab (1:100), and goat anti-CD31 Ab (1:100); 4) washed in PBS, 10 min × 3 times; and, 5) incubated in secondary Abs (diluted in 5% BSA) at RT for 1 h. For primary Abs from rabbit, Alexa Fluor 488-labeled goat anti-rabbit IgG (1:100) were used. Alexa Fluor 488-labeled Donkey anti-goat IgG (1:100) was used for goat primary Ab; 6) washed in PBS, 10 min × 3 times; and, 7) coverslipped using anti-fade mounting medium with or without 10 µg/ml Hoechst dye.
5
Quantifying Infarct Volume and Microvasculature
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Perfused fixed brains were cryopreserved in 25% sucrose and then embedded in OCT and serial cryosectioned. Infarct volume (mm3) was assessed by the Cavalieri Estimator probe using the StereoInvestigator software (MicroBrightField, Williston, VT, USA), as previously described. Briefly, six serial coronal sections, cut at 30 μm, were stained using a 0.2% Cresyl violet solution (Electron Microscopy Science, Hatfield, PA) or anti-mouse IgG-488 (Invitrogen, Waltham, MA). The total volume of infarct was quantified by estimating the area of tissue loss in the ipsilateral cortical hemisphere using six, serial coronal sections. A 100 μm spaced grid was placed over the ipsilateral hemisphere in the Cavalieri probe and infarcted area scored. The coronal sections were used for immunohistochemical (IHC) staining of CD31. Sections were blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO) in 0.2% Triton-X100, incubated with goat anti-CD31 (1 : 100, R&D systems, Minneapolis, MN, USA) overnight at room temperature (RT), washed with 1X PBS, and then incubated with donkey anti-goat 488 (1 : 250, Thermofisher, USA) for 1 hour at RT. Slides were then mounted with DAPI counterstain (SouthernBiotech, Birmingham, AL), and images were acquired using Olympus fluorescence microscope. Microvessel diameters were measured using ImageJ.
6
Immunofluorescent Detection of Antigen Expression
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Antigen expression was confirmed on ice-cold acetone fixed 8 μm cryostat sections of SKRC52 and CT26-CAIX stained with IL2-XE114-TNFmut and IL2-F8-TNFmut (final concentration 5 μg/mL) and detected with rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. IL2-KSF-TNFmut (specific for an irrelevant antigen) was used as negative control. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).
For ex vivo immunofluorescence analysis, mice were injected with 50–60 μg IL2-XE114-TNFmut, IL2-F8-TNFmut, or IL2-KSF-TNFmut and sacrificed 24 h after injection. Organs were excised and embedded in cryo-embedding medium (Thermo Scientific) and cryostat section (10 μm) were stained using the following antibodies: rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).
For ex vivo immunofluorescence analysis, mice were injected with 50–60 μg IL2-XE114-TNFmut, IL2-F8-TNFmut, or IL2-KSF-TNFmut and sacrificed 24 h after injection. Organs were excised and embedded in cryo-embedding medium (Thermo Scientific) and cryostat section (10 μm) were stained using the following antibodies: rat anti-IL2 (eBioscience 14-7029-85) and anti-rat AlexaFluor488 (Invitrogen A21208). For vascular staining goat anti-CD31 (R&D AF3628) and anti-goat AlexaFluor594 (Invitrogen A11058) antibodies were used. Slides were mounted with fluorescent mounting medium and analyzed with Axioskop2 mot plus microscope (Zeiss).
7
Immunofluorescence Analysis of Tumor Immune Landscape
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For immunofluorescence analysis, tumors were embedded in a cryo-embedding medium (NEG-50, Thermo Fisher). Cryostat sections (8–10 μm) were stained using the following primary antibodies: goat anti-CD31 (1:200, R&D Systems; AF3628), rabbit anti-CD4 (1:200, Sino Biological, Wayne, PA; 50,134-R001), rabbit anti-Foxp3 (1:200, Invitrogen; 7000914), rabbit anti-NKp46 (1:200, BioLegend; 137602), and rabbit anti-CD8 (1:200, Sino Biological; 50389-R208). Primary antibodies were detected with anti-rabbit AlexaFluor488 (1:200, Invitrogen; A11008 ) and anti-goat AlexaFluor594 (1:200, Invitrogen; A21209 ). Cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (1:500, Invitrogen; D1306). Slides were mounted with mounting medium (Dako Agilent, Carpinteria, CA) and images were obtained with a Leica DMI6000B confocal microscope (Scientific Center for Optical and Electron Microscopy ScopeM, ETH, Switzerland). Images were analyzed using ImageJ software.
8
Immunohistochemical Labeling of Neural Markers
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Free-floating sections were washed 3 times with PBS (15 min each), blocked for 1 h in blocking buffer (2.5% BSA, 0.3% Triton X-100 PBS), and incubated overnight at 4 °C with the primary antibody in the blocking buffer. The following antisera were used: rabbit anti-AQ-4 (1:500, #AB3594; Millipore, Sigma Aldrich, St. Louis, MO, USA), rabbit anti-YKL-40 (1:300, #PA5-43746; ThermoFisher Scientific, Waltham, MA, USA), goat anti-CD31 (1:500, #AF3628; R&D Systems, Minneapolis, MN, USA), rabbit anti-NeuN (1:1000, #ABN78; Millipore, Sigma-Aldrich, St. Louis, MO, USA), and mouse anti-glial fibrillary acidic protein (GFAP; 1:500, #G3893; Sigma-Aldrich, St. Louis, MO, USA). The next day sections were washed 3 times with the blocking buffer (15 min each), and subsequently incubated for 1 h at room temperature with the secondary antibody in the blocking buffer. The secondary antibodies consisted of donkey anti-rabbit 647 (1:1000, #AB150075; ABCAM, Cambridge, MA, USA), donkey anti-mouse 594, anti-goat 488, and anti-rabbit 488 (each 1:1000, #A21203, #A11055, #A21206, respectively; Invitrogen, and ThermoFisher Scientific, Waltham, MA, USA). Lastly, sections were washed 2 times with PBS (10 min each), and finally mounted onto slides covered with DAPI mounting media (#F6057; Sigma-Aldrich, St. Louis, MO, USA).
9
Immunofluorescent Staining of 4T1 Tumor Sections
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4T1 tissue sections (10 μm thick) were fixed in 4% paraformaldehyde for 10 min, followed by 10 min of permeabilization with 0.1% Triton X-100. Normal donkey serum (10%; Abcam, Cambridge, UK) in PBST (PBS with 0.025% Triton X-100) was used for blocking for 2 hours. Tissue sections were incubated overnight at 4°C in blocking buffer containing rabbit anti–βIII-tubulin (1:1000; Abcam, Cambridge, UK) for neurons detection and goat anti-CD31 (1:200; R&D Systems, Minneapolis) for blood vessels detection. Following overnight incubation, sections were rinsed three times with PBST and then incubated with blocking buffer containing species-appropriate secondary antibodies donkey anti-rabbit Alexa Fluor 488 and donkey anti-goat Alexa Fluor 647 (1:500; Abcam, Cambridge, UK) for 2 hours in RT. Tissue sections were rinsed three times with PBST, then mounted with DAPI-containing Fluoromount (BioLegend, California), coverslipped, and stored in the dark at 4°C until confocal imaging.
10
Immunostaining of Osteocyte Dendrites
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The frozen sections were treated with 0.1% Triton X-100 for 10 minutes and blocked by 3% bovine serum albumin (BSA) for 1 hour. The sections were incubated with goat anti-CD31 (R&D) in 0.2% BSA at 4°C overnight. Then they were incubated with donkey anti-goat secondary antibody in 0.2% BSA at room temperature for 2 hours followed by phalloidin 647 (Invitrogen, 1:250) in PBS at room temperature for 1 hour and DAPI (Servicebio) for 10 minutes. The stained tissue sections were observed and imaged by LSM710 microscope (Zeiss). Osteocytes dendrites were recorded by overexposure of phalloidin.
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