To assess RV histological changes under pressure overload, ventricle tissue was collected from PAB or sham rats at day 3, week 1, week 2, week 4, week 8 post-surgery, respectively. After collection, the heart from each animal were flushed with saline, and then fixed in 10% formaldehyde for 24 h. After paraffin embedding, sections were cut at 5 μm thickness for hematoxylin and eosin (HE) or Masson’s trichrome staining. Cardiomyocyte hypertrophy was assessed by measuring the cross-sectional area of cardiomyocytes, twenty cardiomyocytes were randomly selected from each RV to calculate the mean cross-sectional area of cardiomyocytes. And RV fibrosis was quantitated via calculating the ratios of the fibrosis area to the total area of RV free wall based on Masson’s trichrome staining. The immunofluorescence (IF) staining was performed as previously described [15 (link)]. Briefly, after deparaffinization and antigen retrieval, heart sections were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibody at 4 °C overnight, followed with species-specific Alexa Fluor-coupled secondary antibodies at room temperature for 2 h. The information of primary antibodies is displayed as follows: anti-CD31 (1: 500, GB12063, Servicebio, China), anti-FAP (1: 100, A6349, ABclonal, China). Image analysis was performed using Image J software (NIH, Bethesda, MD, USA).
A6349
Manufactured by ABclonal
Sourced in China
A6349 is a lab equipment product. It is designed for laboratory use. The core function of this product is to assist with certain laboratory procedures. No further details about its intended use or capabilities are available.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd31, by Wuhan Servicebio Technology (1 mentions)
Anti fap, by ABclonal (1 mentions)
Gb12063, by Wuhan Servicebio Technology (1 mentions)
Ecl plus detection system, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fv3000, by Olympus (1 mentions)
Ab150077, by Abcam (1 mentions)
3 protocols using a6349
1
Histological Assessment of Right Ventricular Remodeling
2
Western Blot Protein Detection Protocol
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Western blotting was performed as previously described.9 Briefly, after the quantitation of cell lysates by BCA assay, 5× SDS‐PAGE loading buffer was added and heated at 100°C for 10 min. Next, the protein samples were loaded onto 10% SDS‐PAGE gels for electrophoresis and transferred onto 0.45 μm PVDF membranes. The blots were blocked with 5% nonfat milk powder for 2 h at room temperature, followed by incubation with antibodies against FAP (1:1000, A6349, Abclonal, Wuhan, China) or GAPDH (1:2000, 60 004‐1‐Ig, Proteitech, Wuhan, China) at 4°C overnight. The membrane was then incubated with secondary antibody (1:1000, SA00001‐2, Proteitech) for 1 h, and chemiluminescence signals were detected by an ECL Plus detection system (Thermo Scientific, Waltham, MA, USA).
3
Immunofluorescence and FAP Expression Analysis
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For immunofluorescence detection, NIH3T3 and U87 cells were first fixed with 4% formaldehyde for 20 min and blocked in 5% BSA for 50 min at room temperature. Then, the cells were incubated with anti‐FAP (1:300, A6349, Abclonal, Wuhan, China) and Alexa Fluor 488‐conjugated secondary antibodies (1:500, ab150077, Abcam, Cambridge, MA, USA). The nuclei were counterstained with DAPI (Beyotime), and immunofluorescence images were captured using a laser‐scanning confocal microscope (FV3000, Olympus, Japan).
To measure the protein expression of FAP, NIH3T3 and U87 cells were seeded in 24‐well plates. The cells were incubated with Cy3‐labeled FAP inhibitor (Cy3‐FAPi, Figure5A ) suspended in the cell culture (1 nM) at 37°C for 10 min. Merged brightfield and fluorescent pictures were obtained by confocal microscopy.
To measure the protein expression of FAP, NIH3T3 and U87 cells were seeded in 24‐well plates. The cells were incubated with Cy3‐labeled FAP inhibitor (Cy3‐FAPi, Figure
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