The fixed tissue samples were embedded in paraffin. The paraffin blocks were sliced to a thickness of 4 μm. The EGTA (MVS-0098, Maxim biotechnologies, Fuzhou, China) was used for antigen repair. For histochemistry, F4/80 (28463-1-AP, Proteintech, Wuhan, China), MPO (SKU:023, Biocare Medical, Pacheco, CA, USA), VWF (A0082, DAKO, Glostrup, Denmark), and ITGB3 (MWReg30, BioLegend, San Diego, CA, USA) were used. The sections were incubated with the antibodies at 4 °C overnight. The sections were incubated with anti-rabbit IgG (#7074, Cell Signaling, Danvers, MA, USA) or anti-rat IgG (ab6734, Abcam, Cambridge, UK) for 1.5 h at room temperature and stained with DAB substrate (P0202, Beyotime, Shanghai, China). For immunofluorescence, the sections were incubated with ITGB3 (MWReg30, BioLegend, San Diego, CA, USA) overnight at 4 °C and then incubated with VWF (A0082, DAKO, Glostrup, Denmark) for 2 h at room temperature. For fluorescent staining, the sections were incubated with an Alexa 594-conjugated secondary anti-rat IgG antibody (ab150160, Abcam, Cambridge, UK) and an Alexa 488-conjugated secondary anti-rabbit IgG antibody (ab150077, Abcam, Cambridge, UK). The optical microscope and laser scanning confocal microscope were used to take images. The software ImageJ was used for quantifying the histochemistry staining.
A0082
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom, Germany, Norway
The A0082 is a lab equipment product from Agilent Technologies. It serves as a core functional component for laboratory applications. The detailed specifications and intended use of this product are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
P0226, by Agilent Technologies (7 mentions)
Axio observer z1, by Zeiss (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
M0851, by Agilent Technologies (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
A11008, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Zen 2 blue edition, by Zeiss (2 mentions)
F3520, by Merck Group (2 mentions)
Innovance vwf ac assay, by Siemens (2 mentions)
Ab150077, by Abcam (2 mentions)
Vectastain abc reagent, by Vector Laboratories (2 mentions)
Mab5326, by Merck Group (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Eclipse e600, by Nikon (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Phalloidin tritc, by Merck Group (2 mentions)
Anti rabbit igg, by Cell Signaling Technology (2 mentions)
P8590, by Merck Group (2 mentions)
85 protocols using a0082
1
Histological Examination of Paraffin-Embedded Tissues
2
Enzyme-linked Immunoassay for VWF
3
Quantifying Lung Angiogenesis and Brain Myelination
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We histologically investigated lung angiogenesis and brain myelination by immunostaining. To quantify endothelial density in the lungs, lung sections were stained with von Willebrand factor (vWF) antibody (1:250; A0082, DAKO, Carpentaria, CA, USA) as a primary antibody and stained with Alexa Fluor 568 goat-anti rabbit (1:500; A11036, Invitrogen, Carlsbad, CA, USA) as a secondary antibody. In the brains (cingulate white matter and lateral corpus callosum, -3.60 to -3.08 mm/Bregma), myelinated nerve fibers were stained with myelin basic protein (MBP) antibody [SMI-94] (1:500; ab24567, Abcam, Cambridge, UK) as a primary antibody and Alexa Fluor 488 goat-anti mouse (1:500; A11001, Invitrogen) as a secondary antibody.
4
Immunofluorescence Staining of Vascular Markers
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The sections were deparaffinized in xylene, rehydrated in a graded series of ethanol, and then incubated in 2% bovine serum albumin (BSA, Sigma-Aldrich) in PBS for 30 min at room temperature. The sections were then incubated with the primary antibodies in 2% BSA in a humidified chamber overnight at 4 °C. The following primary antibodies were used: rabbit polyclonal anti-human Von Willebrand factor antibody 1:100 (A0082, Dako, Milan, Italy); mouse monoclonal anti-CD31 antibody 10 μg/mL (ab24590, Abcam, Cambridge, UK); mouse monoclonal anti-FLK-1 antibody 1:100 (sc-6251, Santa Cruz Biotechnology Inc., Paso Robles, CA, USA); and mouse monoclonal anti-OB antibody 1:100 (sc-28344, Santa Cruz Biotechnology, Inc.). Immunofluorescence staining was performed for 1 h at room temperature with the secondary antibody DyLight 488-labeled anti-mouse IgG (KPL, Gaithersburg, MD, USA) or DyLight 549-labeled anti-rabbit IgG (H + L) (KPL) diluted to 1:1000 in 2% BSA. Nuclear staining was performed with 2 µg/mL Hoechst H33342 (Sigma-Aldrich) for 2 min. The sections were coverslipped with a drop of mounting medium.
5
Immunofluorescence Analysis of Tissue Markers
6
Immunohistochemical Analysis of Vascular and Immune Markers
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The immunohistochemistry was performed as reported earlier (Sethi 2013). Briefly tissue sections were de-paraffinized and rehydrated. The tissue peroxidases were inactivated with 0.5 % H2O2 in methanol for 20 min. Pepsin (2 mg/ml in 0.01 N HCl) was used to unmask antigen-binding sites (60 min) and then 1 % bovine serum albumin (BSA) in PBS was used to prevent non-specific binding (30 min). Next the tissues were incubated overnight (16 h) at 4 °C with the following antibodies: von Willebrand Factor (1:500, DAKO A0082), Toll-Like Receptor 4 (1:25, IMG-578A, IMGENEX) and Toll-Like Receptor 9 (1:50, IMG-3051, IMGENEX) followed by appropriate secondary antibody (vWF at 1:300 and TLR at 1:100, all from DAKO). VECTOR VIP Peroxidase Substrate Kit (Vector laboratories, Burlingame, CA) was used for colour-development ed followed by counter staining with methyl green (Vector laboratories).
7
Integrin and VWF Western Blot
8
Multimeric Analysis of VWF and ADAMTS13
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Analysis of multimeric VWF was carried out as described28 using a polyclonal rabbit anti‐human VWF antibody (A0082; Dako) in combination with an alkaline phosphatase–conjugated polyclonal anti‐rabbit antibody (Promega) for detection of VWF. Multimeric ADAMTS13 was similarly analyzed by vertical agarose gel electrophoresis under nonreducing conditions. Appropriately diluted ADAMTS13 samples (22.5 μL) were mixed with 9 μL of a 750 mmol/L IAA solution and incubated for 10 minutes in darkness at RT. Following the addition of 31.5 μL sample buffer (70 mmol/L Tris, 4 mmol/L EDTA, 24 g/L sodium dodecyl sulfate [SDS], 9 mol/L urea, 0.01 g/L bromophenol blue; pH 6.7), samples were incubated for 1 hour at 37°C before loading onto the agarose gel. After blotting, ADAMTS13 was detected with a polyclonal rabbit anti‐ADAMTS13 antibody (K1‐4; Baxter) in combination with an alkaline phosphatase–conjugated polyclonal anti‐rabbit antibody (Promega).
9
Immunohistochemical Analysis of Lung and Liver
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Formalin-fixed paraffin-embedded lung sections were stained with Alcian blue Elastin Van Gieson and immunostained for αSMA (α-smooth muscle actin; 1:150; Dako, M0851), von Willebrand factor (1:300; Dako, A0082), NF-κB (nuclear factor kappa-B; 1:400; Cell Signaling, D14E12), F4/80 (1:100; Abcam, ab111101), iNOS (inducible NO synthase; 1:100; Abcam, ab15323), and CD206 (1:100; R&D Systems, AF2535). As isotype control, IgG and IgG2a were used. Formalin-fixed paraffin-embedded liver sections were stained with anti-F4/80 to confirm macrophage ablation. For immunohistochemical and immunofluorescent staining, a standard protocol was followed. Histological images were visualized using a Zeiss multislide scanning microscope (Imager.Z2; Carl Zeiss, Ltd) with an Axiocam 506 color camera (Zeiss) for immunohistochemical images and MRm camera (Zeiss) for immunofluorescent images. Slides were scanned sequentially using ×20 magnification objective lens, and the analysis was performed in Zen2 blue edition (Zeiss). For all tissue macrophage quantification, positively stained cells were counted in 6 fields of view at ×200 magnification and normalized to the control group.
10
Bone Sample Immunohistochemistry Staining
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