After collection, tissues were fixed in 10% neutral buffered formalin (NBF) and processed for paraffin embedment. Sections from each tissue block were counterstained with hematoxylin/eosin (H&E) and analyzed for antigen detection: iNOS (NB300-605, Novus Biologicals), IL-19 (PA5-68455, Invitrogen), COX-2 (27308-1-AP, Proteintech), 3-chloro-tyrosine epitopes (HP5002, HycultBiotech, Uden, Netherlands), and Ki67 (ab15580, Abcam) following our published procedures [41 ,42 (link)]. In brief, following deparaffinization and hydration, slides were subjected to heated antigen retrieval (citric, pH 6.0 or Tris-EDTA, pH 9.0). After overnight incubation in primary antibody, slides were incubated with biotinylated anti-mouse/anti-rabbit secondary antibody (RTU PK7200, Vector Laboratories, Burlingame, CA, USA) and a subsequent incubation with avidin/biotin streptavidin/horseradish peroxidase (Vectastain ABC, SK-4103, Vector Laboratories). Then, slides were developed with a diaminobenzidine/hydrogen peroxide mixture, counterstained with hematoxylin, dehydrated with graded alcohols and xylene, and mounted using a xylene based medium. Images were captured using an Olympus BX50 and Spot (Model 2.3.0) camera.
Hp5002
Manufactured by Hycult Biotech
Sourced in Netherlands
The HP5002 is a compact and high-performance microplate reader designed for a wide range of immunoassay and cell-based applications. It features a wavelength range of 340-850 nm and supports multiple detection modes, including absorbance, fluorescence, and luminescence. The instrument is equipped with a temperature-controlled incubation chamber and shaking capabilities to ensure optimal sample handling and consistent results. The HP5002 is a versatile and reliable tool for researchers and laboratories requiring accurate and efficient microplate-based analysis.
Automatically generated - may contain errors
Lab products found in correlation
Gel documentation system, by Bio-Rad (2 mentions)
Ab7817, by Abcam (2 mentions)
Ab3471, by Abcam (2 mentions)
Abs1675, by Merck Group (2 mentions)
Ab37647, by Abcam (2 mentions)
Ab15580, by Abcam (1 mentions)
Rtu pk7200, by Vector Laboratories (1 mentions)
Pv 9001, by ZSGB-BIO (1 mentions)
Pa5 16672, by Thermo Fisher Scientific (1 mentions)
Ab11861, by Abcam (1 mentions)
Ab55811, by Abcam (1 mentions)
Pv 9003, by ZSGB-BIO (1 mentions)
Af2655, by R&D Systems (1 mentions)
Pv 9004, by ZSGB-BIO (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
P smad2 3, by Cell Signaling Technology (1 mentions)
6 protocols using hp5002
1
Tissue Antigen Detection Protocol
2
Immunohistochemical Analysis of Liver Sections
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Fresh-frozen liver sections were incubated with antibody to C1q (ab11861, Abcam, 1:50 dilution), and formalin-fixed, paraffin embedded liver sections were incubated as described (23 (link)) with antibodies to C3d (AF2655, R&D, 1:500 dilution), MAC (ab55811, Abcam, 1:800 dilution), C1q (ab11861, Abcam, 1:50 dilution), MPO (PA5-16672, Thermo Fisher, 1:400 dilution), 3-chlorotyrosine (HP5002, Hycult Biotech, 1:200 dilution), F4/80 (70076s, CST, 1:500 dilution), and VCAM-1 (MA5- 31965, Thermo Fisher, 1:400 dilution), followed by incubation with secondary antibody (HRP-polymer detection kit PV-9001, PV-9003 or PV-9004, Zsbio), according to the manufacturer’s instructions. Immunohistochemistry results were graded based on the staining intensity and percent positive cells as described (24 (link)).
3
Cardiac Fibroblast Protein Expression Analysis
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Lysates of cultured cardiac fibroblasts and tissues lysates of infarcted hearts were extracted using a lysis buffer (Beyotime, Shanghai, China) with a protease inhibitor cocktail (Roche, Germany). Protein blotting was performed with a modified standard protocol using primary antibodies to VPO1 (1:1000, ABS1675, Millipore, USA), α-SMA (1:1000; ab7817, Abcam, UK), collagen I (1:1000; ab3471, Abcam, UK), smad2/3 (#8685), P-smad2/3 (#9510), ERK1/2 (#4695), P-ERK1/2 (#4370) (1:1000; Cell Signaling Technology, USA), 3-chlorotyrosine (1:500; HP5002, Hycult Biotech, Netherlands), GAPDH (1:5000, G8795, Sigma-Aldrich, USA) overnight at 4 °C. PVDF membranes were incubated with HRP--conjugated secondary antibodies and bands were visualized by a gel documentation system (Bio-Rad, USA).
4
Oxidative Stress Signaling in Aorta and HUVEC
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Isolated thoracic aortic tissues or cultured HUVECs were lysed in commercially purchased RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) spiked with 1 mmol/L phenylmethanesulfonyl fluoride (ST506, Beyotime, Shanghai, China) to obtain protein products. The antibodies against the following proteins were used: PXDN (1 μg/mL, ABS1675, Merck, Frankfurter, Germany), RAGE (0.3 μg/mL, ab37647, Abcam, Cambridge, UK), NOX2 (0.25 μg/mL, ab80508, Abcam, Cambridge, UK), NOX1 (0.5 μg/mL, ab131088 and ab121009, Abcam, Cambridge, UK), NOX4 (0.33 μg/mL, ab154244, Abcam, Cambridge, UK), 3-chlorotyrosine (0.1 μg/mL, 3-Cl-Tyr, HP5002, Hycult biotech, Uden, Netherlands), 4-hydroxynonenal (0.43 μg/mL, 4-HNE, ab46545, Abcam, Cambridge, UK), Akt (0.5 μg/mL, SAB4500797, Sigma-Aldrich, USA), p-Akt (0.143 μg/mL, ab18206, Abcam, Cambridge, UK), eNOS (0.25 μg/mL, 61029, BD, Biosciences, NJ, USA) and p-eNOS (0.056 μg/mL dilution, Ser1177, MA5-14957, Invitrogen, CA, USA). Expression of the protein GAPDH (0.5 μg/mL, SAB1405848, Sigma-Aldrich, MO, USA) was used for data normalization. PVDF membranes were incubated with a horseradish peroxidase linked secondary antibody and bands were visualized using gel documentation system (Bio-Rad).
5
Quantifying Cardiac Infarct and Fibrosis
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Human and mouse heart tissues were fixed overnight in 4% paraformaldehyde. Samples were embedded in paraffin wax and sliced into 4 μm sections. Masson trichrome stain (Sigma-Aldrich, USA) was used according to the manufacture's protocol. Infarct size, expressed as a percentage, was calculated by dividing the sum of infarct areas from all sections by the sum of LV areas from all sections (including those without infarct scar) and multiplying by 100 [14 (link)]. Infarct expansion index was calculated using the following formula: expansion index = (LV cavity area/total heart area) × (uninfarcted septal thickness/infarcted LV free wall thickness) [15 ]. Rabbit anti-VPO1 (1:200; ABS1675, Millipore, USA), anti-3-chlorotyrosine (1:100; HP5002, Hycult Biotech, Netherlands), anti-collagen I (1:200; ab3471, Abcam, UK), anti-ki67 (1:200, ab16667, Abcam, UK), and mouse anti-α-SMA antibody (1:200; ab7817, Abcam, UK) were used as primary antibodies for immunochemical staining, followed by secondary antibodies (1:100; ZSGB-BIO, China) and DAB reagents. Quantification of fibrosis, VPO1, α-SMA and collagen I in heart tissues was determined by Imag-Pro-Plus 6.0 software.
6
Immunohistochemical Analysis of Vascular Markers
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Cryosection of thoracic aortas and mesenteric arteries were blocked in 5% bovine serum albumin (BSA, A1933, Sigma-Aldrich, MO, USA) diluted in phosphate buffered saline (PBS) for 1 h at room temperature and subsequently incubated with goat anti-PXDN (10 μg/mL, sc-168598, Santa Cruz, TX, USA) and rabbit anti-CD31 (1:20 dilution, ab28364, Abcam, Cambridge, UK), anti-RAGE (1 μg/mL, ab37647, Abcam, Cambridge, UK) and anti-3chlorotyrosine (2 μg/mL, HP5002, Hycult biotech, Uden, Netherlands) antibodies overnight at 4 °C in a humidity box. Samples were then incubated with secondary antibody for 1 h at room temperature. Finally, DAPI (5 μg/mL, D9542, Sigma-Aldrich, MO, USA) was used for nuclear staining.
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