HAECs were cultured on glass coverslips in a 24-well plate and fixed with 4% paraformaldehyde for 10 min. Then, the cells were permeabilized with 0.25% Triton X-100 and blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature. Subsequently, the cells were incubated with primary antibodies against TXNIP (1:100; Proteintech) and TRX (1:200; Proteintech) overnight at 4 °C. Alexa Fluor 555-labeled and FITC-labeled secondary antibodies (all from Beyotime, Shanghai, China) were used to detect TXNIP (in red) and TRX (in green), respectively. DAPI was then added for nuclear staining. Fluorescence signals were captured under a confocal microscope (Carl Zeiss, Oberkochen, Germany) and quantitatively analyzed using Image-Pro Plus software.
Image pro plus software
Manufactured by Zeiss
Sourced in Germany
Image-Pro Plus software is a powerful image analysis and processing tool developed by Zeiss. It provides a comprehensive set of functions for capturing, processing, analyzing, and reporting images. The software supports a wide range of image file formats and offers advanced features for image enhancement, measurement, and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Clsm software, by Zeiss (3 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Fitc labeled secondary antibody, by Beyotime (1 mentions)
880 airyscan, by Zeiss (1 mentions)
Axio imager a2, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Calcein am, by Genmed Scientifics (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Standard light microscopy, by Zeiss (1 mentions)
Vectastain abc system, by Vector Laboratories (1 mentions)
Anti osteocalcin, by Merck Group (1 mentions)
Anti vegf, by Abcam (1 mentions)
7 protocols using image pro plus software
1
Immunofluorescence Imaging of TXNIP and TRX
2
Immunofluorescence Labeling of Oocytes
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Oocytes were fixed in 2% paraformaldehyde (PFA) in PEM buffer (100 mM Pipes, pH 6.9, 1 mM MgCl2, 1 mM EGTA) with 0.5% Triton X‐100 for 1 h at room temperature, then washed and blocked for 1 h in phosphate buffer saline (PBS) added with 10% normal goat serum. The blocked oocytes were incubated in blocking buffer with primary antibodies at 4°C overnight. Antibodies used in the experiments are described in Table S1 . After being washed three times (5 min each) in PBS containing 0.2% Triton X‐100, oocytes were labelled with appropriate secondary antibodies for 1 h at room temperature, then washed and mounted on glass slides in mountain medium with DAPI (Vector laboratories). The fluorescent signals from both control and experimental oocytes were acquired by setting up the same parameters of the upright fluorescent microscope (ZEISS Axio Imager A2) or confocal microscope(ZEISS 880 Airyscan) semi‐quantitative analysed by ImagePro Plus software and Zeiss analysis software.
3
Quantifying Intracellular ROS via DCF Fluorescence
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The fluorescence intensity of 2′,7′-dichlorofluorescein (DCF; Molecular Probes, Eugene, OR, USA) was used to determine intracellular ROS generation. Within the cell, esterases cleaved the acetate groups on 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Applygen Technologies Co., Ltd, Beijing, People’s Republic of China), intracellularly trapping the reduced probe dichlorodihydrofluorescein (DCFH). ROS in the cells would then rapidly oxidize DCFH, yielding the highly fluorescent product DCF. Six hours after SDT, the cells were washed twice with PBS and incubated with DCFH-DA (20 μM, diluted in serum-free medium) for 30 minutes at 37°C in the dark. Then, the cells were carefully washed twice with PBS. Immediately after washing, fluorescence was measured via CLSM at 488 nm excitation and 525 nm emission wavelengths. The obtained images were subsequently processed using Image-Pro Plus software and Zeiss CLSM software.
4
Mitochondrial Calcein Staining and mPTP Imaging
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Mitochondria were stained with the acetomethoxy derivate of calcein (calcein–AM) through Co2+ quenching of cytosolic calcein fluorescence. Six hours after SDT, macrophages were loaded with 5 μM calcein–AM (GenMed Scientifics Inc., Wilmington, DE, USA) in the presence of 5 mM cobalt chloride in the dark for 20 minutes at 37°C as previously described.29 (link) The cells were carefully washed twice with PBS, and fluorescence was monitored via fluorescence microscopy. calcein–AM fluorescence was measured at 525 nm following excitation at 488 nm. To investigate the mechanism of mPTP opening, the cells were pretreated with NAC and inhibitors of the different subunits (BA, DIDS, and CsA) detected through CLSM. Images were subsequently processed using Image-Pro Plus software and Zeiss CLSM software.
5
Cardiomyocyte Size Measurement via Histology
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Heart Sects. (5 µm) were examined by HE staining (Service Biological Technology Co., Ltd, Wuhan, China) according to the provided procedures. The images were observed under a light microscope (Olympus Corporation, Tokyo, Japan), and were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Bethesda, MD, USA).
Heart Sects. (5 µm) were stained using WGA (Invitrogen Inc., CA, USA) to measure the cross-sectional area of cardiomyocytes. The images were observed under a confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany), and were analyzed with Image-Pro Plus software. Briefly, cardiomyocyte size was evaluated by measuring the cross-sectional area along the short axis of cardiomyocytes.
Heart Sects. (5 µm) were stained using WGA (Invitrogen Inc., CA, USA) to measure the cross-sectional area of cardiomyocytes. The images were observed under a confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany), and were analyzed with Image-Pro Plus software. Briefly, cardiomyocyte size was evaluated by measuring the cross-sectional area along the short axis of cardiomyocytes.
6
Histological Analysis of Mouse Bone Tissue
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Tissues were fixed in 4% paraformaldehyde and decalcified in 10% EDTA. Paraffin sections were stained with Masson’s trichrome stain for morphological analysis. For IHC, antigens of de-paraffinized sections were retrieved by 0.05% trypsin or hyaluronidase (10 mg/ml) and treated with 3% H2O2. After blocking with 5% normal goat serum, tissues were incubated with primary antibodies in 4°C, overnight. The following rabbit polyclonal antibodies against mouse were used, anti-VEGF (Abcam, Cambridge, UK), anti-PECAM (Abcam), and anti-Osteocalcin (Millipore, Billerica, MA, USA). Sections were then incubated with anti-rabbit secondary antibody (Vectastain ABC system, Vector Laboratories, Servion, Switzerland) and developed with 0.1% 3, 39-diaminobenzidine. Images were captured using standard light microscopy (Zeiss, Oberkochen, Germany) and quantified using Image-Pro Plus software (Rockville, MD, USA). Data from three independent mice staining were used for statistical analysis.
7
Intracellular ROS Measurement using DCF
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The florescence intensity of 2′, 7′-dichloroflorescein (DCF; Molecular Probes, Eugene, OR, USA) was used to determine intracellular ROS generation. Within the cell, esterase cleaved the acetate groups on 2′, 7′dichlorofluorescein diacetate (DCFH-DA; Applygen Technologies Co., Ltd, Beijing, China), intracellularly trapping the reduced probe dichlorodihydroflorescein (DCFH). ROS in the cells would then rapidly oxidize DCFH, yielding the highly florescent product DCF. Six h after SDT, the cells were washed twice with PBS and incubated with DCFH-DA (20 µM, diluted in serum-free medium) for 30 min at 37°C in the dark. Then, the cells were carefully washed twice with PBS. Immediately after washing, florescence was measured via CLSM (LSM 510 Meta; Zeiss, Gottingen, Germany) at 488 nm excitation and 525 nm emission wavelengths. The obtained images were subsequently processed using Image-Pro Plus software and Zeiss CLSM software.
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