Human VWF was purchased from Merck Chemicals; histamine from Sigma Aldrich, Germany. The recombinant VWF domains (A1, A2, and A3) were produced in HEK293 cells (Posch et al., 2017 (link)). Pneumococcus-specific polyclonal antiserum was generated in rabbit against heat inactivated serotype 35A and 2 bacteria (Pineda, Germany). IgG was purified using protein A/G sepharose. Mouse anti-human VWF antibody (polyclonal IgG) and FITC-conjugated VWF-specific antibody were purchased from Abcam (ab8822), peroxidase-conjugated mouse anti-VWF antibody from Hämochrom Diagnostika; Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse IgG, Alexa Fluor 568-conjugated goat anti-rabbit and goat anti-mouse IgG were from Thermo Fisher Scientific (formerly Invitrogen), paraformaldehyde (PFA) was purchased from EM Science and mounting medium from Dako.
Ab8822
Manufactured by Abcam
Sourced in United Kingdom
Ab8822 is a monoclonal antibody that recognizes an epitope located within the C-terminus of the human Caspase-3 protein. The antibody can be used for the detection of Caspase-3 in various applications, such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab5694, by Abcam (3 mentions)
Lsm 700, by Zeiss (3 mentions)
Ab119339, by Abcam (2 mentions)
Ab185732, by Abcam (2 mentions)
Ab6586, by Abcam (2 mentions)
Ab150068, by Abcam (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Lsm 710, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Histamine, by Merck Group (1 mentions)
C6198, by Merck Group (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
Alpha smooth muscle actin, by Merck Group (1 mentions)
Vector antigen unmasking solution, by Vector Laboratories (1 mentions)
Bs 1278r, by Bioss Antibodies (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Ab175473, by Abcam (1 mentions)
Ab6308, by Abcam (1 mentions)
14 protocols using ab8822
1
Recombinant von Willebrand Factor Domains Protocol
2
Visualizing Aortic Endothelial Cells
3
Quantifying Vessel Occlusion in PAH Lungs
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Immunofluorescent staining with von Willebrand factor antibody conjugated to FITC (vWf, 1:200 dilution, overnight incubation at 4°C, Abcam, Ab8822) combined with α‐smooth muscle actin conjugated to Cy3 (α‐SMA, 1:200 dilution, 2 h at room temperature, Sigma, C6198) was performed on control and PAH lung tissue to determine the number of occluded vessels as percentage of the total number of vessels. Approximately, 30–68 vessels below 100 μm were studied in 7–12 fields of view per subject. Occlusion was defined by vWf staining completely comprising the luminal surface of a vessel. Imaging was performed at 10× magnification with an Axiovert 200 Marianas inverted wide‐field fluorescence microscope (Carl Zeiss Microscopy, Jena, Germany). These percentages were averaged for controls and patients and given as mean ± SEM.
4
Quantifying Mature Vessel Density Post-MI
5
Oxidative Damage in Rat Lungs
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Rat lungs were collected and inflated with an 1% solution of low-melt-agarose and fixed in formalin and embedded in paraffin. Five micrometer-thick lung paraffin sections were deparaffinized and rehydrated. Sections were boiled for 40 min in Vector® Antigen Unmasking Solution (Vector, Burlingame, USA) using a pressure cooker. After blocking with goat serum 10% (ThermoFisher Scientific, Massachusetts, USA), sections were incubated overnight at 4 °C with primary antibodies directed against 8-OHdG (1:150; bs-1278R, Bioss Antibodies, Woburn, MA, USA), co-stained with von Willebrand factor (1:1000; ab8822, Abcam, Cambridge, UK), and alpha smooth muscle actin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). All sections were mounted with ProLong® Gold antifade reagent (Invitrogen, Massachusetts, USA) containing DAPI.
6
Immunofluorescence Staining of Tissue Sections
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Sections (8 μm thickness) were cut using the cryostat (Microm HM 525, Thermo Scientific). Immunofluorescence staining was performed using the following primary antibodies: mouse anti-CD31 (ab119339, Abcam, Cambridge, UK), rabbit anti-CD34 (ab185732, Abcam), mouse anti-collagen type I (ab6308, Abcam), rabbit anti-collagen type IV (ab6586, Abcam) or FITC-conjugated von Willebrand factor (ab8822, Abcam). The following secondary antibodies were used: goat anti-mouse Alexa Fluor 568 conjugate (ab175473, Abcam) and donkey anti-rabbit Alexa Fluor 488 conjugate (ab150073, Abcam). Autofluorescence was minimized with the Autofluorescence Eliminator Reagent (Millipore) according to the manufacturer’s instructions. All sections were counterstained with DAPI (Sigma) and mounted with ProLong Gold Antifade (Life Technologies, Carlsbad, CA, USA). Staining was visualized by confocal microscopy (LSM 700, Carl Zeiss, Oberkochen, Germany).
7
Flow Cytometry Analysis of Endothelial Markers
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Surface expression of CXCR7, Flk-1, Flt-1, vWF, VE-cadherin, and CD31 was evaluated by flow cytometric analysis. Cells were harvested with PBS containing 5 mM EDTA and immediately neutralized in FACS buffer (α-MEM containing 1% BSA and 0.025% NaN3). After extensive washing with FACS buffer, cells (105 cells) were incubated with 1 µg/ml of the primary antibodies, including CXCR7 (MAB42273, R&D Systems, 1:100 dilution), Flk-1 (Avas12a1, Novus, 1:100 dilution), Flt-1 (MAB321, R&D System, 1:100 dilution), vWF (ab8822, Abcam, 1:100 dilution), VE-cadherin (FAB9381P, R&D System, 1:100 dilution), and CD31 (FAB806G, R&D Systems, 1:100 dilution) by shaking for 1 h at 4 °C. After extensive washing with FACS buffer, cells were incubated with DyLight 649 AffiniPure goat anti-rabbit IgG or DyLight 488 AffiniPure goat anti-mouse IgG (115-495-209 or 111-545-144, Jackson Immunoresearch, 1:100 dilutions) by shaking for 1 h at 4 °C. Cells were then washed with FACS buffer five to six times, and fixed in PBS containing 1% paraformaldehyde. Expression levels were measured on a FACScalibur instrument and FACSDiva 6.0 software (BD Bioscience).
8
Quantifying Myocardial Angiogenesis Post-Infarction
9
Immunohistochemical Analysis of Rat Lung Tissue
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Rat lung tissues were fixed with 10% neutral buffered formalin (NBF) for 24 h, then embedded in paraffin. The paraffin blocks were sectioned at 5 μm and deparaffinized using Histoclear (Chayon, Seoul, Republic of Korea, HS-200). After rehydration, antigen retrieval was performed with Borg Decloaker RTU solution (Biocare Medical, Pacheco, CA, USA, BRR1000AG1) for 15 min in a high-pressure cooker and then blocked in blocking buffer (1% BSA, 5% goat serum, 1xPBS) for 1 h at room temperature. The sections were incubated with anti-POSTN (1:200, Abcam, ab14041), Von Willebrand Factor (vWF)-FITC (1:200, Abcam, ab8822) overnight at 4 °C. The following day, the sections were incubated with secondary antibody Alexa Fluor secondary rabbit 568 for 1 h at room temperature and counterstained with 4’,6-Diamidino-2-Phenylindole (DAPI) (Thermo Fisher Scientific) to visualize the nuclei. Imaging was performed using a Zeiss confocal microscope (ZEISS LSM-700, Carl Zeiss, Oberkochen, Germany).
10
Immunostaining of Vascular and Cardiac Cells
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Spiral and straight vessels, after culture for the designated time, were fixed by treatment with 4% formaldehyde for 15 min (for PDMS devices) or 60 min (for collagen devices). Devices were permeabilized by incubation in phosphate-buffered saline (PBS) containing Triton X-100 (0.5%) and bovine serum albumin (BSA; 2%) for 30 min. Unconjugated primary antibodies were added into the spirals (and onto collagen gels), incubated overnight at 4°C, followed by washing with PBS for three times (5 min for each time), and incubated with secondary antibodies and Hoechst 33342 (40 μg/ml) for 1 hour at room temperature before a final round of washing. Devices were stained for factors including CD31 (1:30; Abcam ab28364), VE-cadherin (1:100; Abcam ab33168), von Willebrand factor (1:100; Abcam ab8822), cTnT (cardiac troponin T) (1:100; Thermo Fisher Scientific MS-295-P1), and F-actin (1:100; Thermo Fisher Scientific A12379). Stained devices were imaged using a Nikon A1R confocal microscope or a Yokogawa W1 spinning disc confocal system.
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