Hematoxylin and Eosin (H&E) staining was done according to the previously described method [17 (link),18 ]. The slides were incubated with Harris hematoxylin for 10 seconds, washed, treated with eosin (Sigma Aldrich Co., St. Louis, MO, USA) for 5 seconds, and washed again. After drying, the slides were dehydrated by soaking in ethanol and xylene, and sealed with Permount (Thermo Fisher Scientific). Images were captured using an Image Analysis System (Leica Microsystems, Wetzlar, Germany).
Image analysis system
Manufactured by Leica
Sourced in Germany, Spain
The Image analysis system is a software tool designed to capture, process, and analyze digital images. It provides advanced image processing capabilities, enabling users to perform tasks such as image enhancement, segmentation, and quantitative analysis. The core function of this system is to facilitate the extraction of meaningful information from digital images, supporting research and analysis across various fields.
Automatically generated - may contain errors
Lab products found in correlation
Sirius red f3ba, by Merck Group (2 mentions)
Ix 70, by Olympus (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Pore polyester membrane insert, by Corning (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Rabbit anti ki67, by Proteintech (1 mentions)
Rabbit anti dec2, by Proteintech (1 mentions)
Biotinylated goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Optical microscope, by Leica (1 mentions)
Rm2235 rotary microtome, by Leica (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Ab53444, by Abcam (1 mentions)
Masson s trichrome staining, by Merck Group (1 mentions)
Dmre microscope, by Leica (1 mentions)
Shandon zinc formal fixx, by Thermo Fisher Scientific (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
19 protocols using image analysis system
1
Hematoxylin and Eosin Staining Protocol
2
Collagen Gel Contraction Assay with Lipoaspirate
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To determinate the effect of lipoaspirate on matrix contraction, a collagen gel (3.5 mg/mL) was obtained from rat tail tendons by acetic acid extraction [25 (link)]. Fifty microliters of collagen were added to 50 μL of fibroblasts (1 × 105 cells/mL) in serum-free basal media prior to gel formation, and the solution was allowed to polymerize for 30–45 min at 37 °C. The solidified gels were gently detached from the plastic surface to allow contraction. The gels were then incubated at 37 °C in 5% CO2 in 1 mL of serum-deprived medium treated with or without the tested stimuli:
Blank: collagen with culture medium (without cells and stimuli);
Ctr: collagen with cells without stimuli;
Ctr+: collagen with cells + TGFβ1 5 ng/mL;
LIPO: collagen with cells + 15% v/v of LipoCM.
3
Transwell Invasion Assay for CD63 Modulation
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The Transwell invasion assay was performed to examine the invasion ability of CD63-silenced and CD63-overexpressing TCA8113 cells, using a 6.5-mm Transwell with an 8.0-µm Pore Polyester Membrane Insert (Corning Incorporated) coated with 10 µl Matrigel (50 µl/cm2; Corning Incorporated). A total of 1×105 cells were plated into the upper chamber of the Transwell with 500 µl RPMI 1640 medium without FBS, and 500 µl RPMI 1640 medium with 10% FBS was added into the lower chamber. The cells were cultured for 24 h in 5% CO2 at 37°C.
The non-invading cells in the upper side of the filter were then gently removed with a soft cotton swab, and the cells that had invaded to the lower side of the filter were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% crystal violet at 37°C for 15 min (Sigma-Aldrich; Merck KGaA). The number of cells in three randomly selected fields was counted with an Image Analysis System (version 3.3.0; Leica Microsystems GmbH), and these numbers are expressed as the average number of migrating cells.
The non-invading cells in the upper side of the filter were then gently removed with a soft cotton swab, and the cells that had invaded to the lower side of the filter were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% crystal violet at 37°C for 15 min (Sigma-Aldrich; Merck KGaA). The number of cells in three randomly selected fields was counted with an Image Analysis System (version 3.3.0; Leica Microsystems GmbH), and these numbers are expressed as the average number of migrating cells.
4
Cardiac Collagen Quantification Protocol
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A piece of LV from each rat was fixed in 3, 7% paraformaldehyde for 24 hours, and then stored in 70% alcohol until its use. The tissues were dehydrated, and fixed in paraffin and then cut into 4 µm slices. These slices were stained with Sirius Red F3BA (0.5% in saturated aqueous picric acid; Aldrich Chemical Company, Madrid, Spain). Quantification of collagen content was performed using an image analysis system (Leica Microsystems, Barcelona, Spain). The analyses were performed in four different sections of each slide of the heart and ten photographs from each section were taken. A single investigator, unaware of the experimental groups, performed these analyses.
5
Quantifying Cardiac Calcium Content
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To assess calcium content, paraffin-embedded heart 4-mm slices were stained with alizarin red (acid; Aldrich Chemical Company, Madrid, Spain). Four different sections of each slide of the heart and ten photographs from each section were taken using an image analysis system (Leica Microsystems, Barcelona, Spain). A single investigator, blinded to the nature of the samples, performed the analyses.
6
Quantifying Renal Collagen Content
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To asses renal collagen content, paraffin-embedded kidneys were cut into 4-mm slices and stained with Sirius Red F3BA (0.5% in saturated aqueous picric acid; Aldrich Chemical Company, Madrid, Spain). Four different sections of each slide of the kidney and ten photographs from each section were taken using an image analysis system (Leica Microsystems, Barcelona, Spain). A single investigator, blinded to the nature of the samples, performed the analyses.
7
Lymphocyte Karyotyping by G-Banding
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2 mL of peripheral blood was collected under sterile conditions, which, after being anticoagulated with heparin, was inoculated into lymphocyte medium and then cultured at 37°C for 72 h to collect cells, followed by a G-banding process for analysis using the image analysis system (Leica, United States).
8
Quantifying Vascular Re-endothelialization Post-Injury
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Re-endothelialization was quantified in Evans blue-stained carotid arteries at 7, 14 and 21 days after vascular injury. Evans blue was injected into the injured common carotid artery of rats at the dosage of 25 mg/kg through tail vein. Thirty minutesafter the Evans blue injection, the rats were anesthetized with 2% pentobarbital sodium solution at the dosage of 75 mg/kg.
Rats were sacrificed by removing the common carotid artery and cultured in PBS. The common carotid arteries were placed into a petri dish containing PBS and were cut longitudinally by removing excess muscles and connective tissue. These arteries were rinsed again with PBS and then plated on glass slide. Images were captured using a micro-camera to determine the re-endothelialization of the injured vessels. The total area of the vessels and the area of the Evans blue stained area were measured with Lecia Qwin image analysis system. The percentage of re-endothelialization of the injured vessels was calculated by the following equation: (the total area of injured blood vessel - the area of the Evans blue stained area)/the total area of the injured blood vessel.
Rats were sacrificed by removing the common carotid artery and cultured in PBS. The common carotid arteries were placed into a petri dish containing PBS and were cut longitudinally by removing excess muscles and connective tissue. These arteries were rinsed again with PBS and then plated on glass slide. Images were captured using a micro-camera to determine the re-endothelialization of the injured vessels. The total area of the vessels and the area of the Evans blue stained area were measured with Lecia Qwin image analysis system. The percentage of re-endothelialization of the injured vessels was calculated by the following equation: (the total area of injured blood vessel - the area of the Evans blue stained area)/the total area of the injured blood vessel.
9
Immunohistochemical Analysis of DEC2, Ki-67, and NR2F1
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Paraffin-embedded sections were cut into 4um and deparaffinized in xylene and rehydrated, and endogenous peroxidase was blocked with 3%H2O2. Antigen retrieval was accomplished by 0.01 mol/L citrate buffer solution (pH 6.0) in a 700 W microwave oven for 15 min. After incubation with 5% normal goat serum for 20 min, the slides were exposed overnight at 4 °C to the rabbit anti-DEC2 (1:150; Proteintech), rabbit anti-Ki-67(1:800; Proteintech), rabbit anti-NR2F1 (1:200; Proteintech). Sections were then incubated with biotinylated goat anti-rabbit IgG (Zhongshan Goldenbridge Biotechnology) for 1 h, and streptavidin-peroxidase for 30 min. The 0.02% diaminobenzidine tetrahydrochloride was used as a chromogen, and the slides were counterstained with hematoxylin. The percentage of positive cells was estimated using an image analysis system (Leica).
10
Immunohistochemical Analysis of Collagen I and III
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A manual procedure was performed according to instructions of the SP-9002 HistostainTM-Plus kit (ZYMED, CA, USA). Sections were deparaffinized, rehydrated and immersed in 10 mM citric acid (pH 6.0) to exclude epitope masking due to fixation. Sections were immunostained with primary antibodies against COL I (1:50 dilution, BA0325, Boster Bioengineering Co. Ltd., Wuhan, China) and COL III (1:50 dilution, BA0326, Boster Bioengineering Co. Ltd.) at 4°C overnight. Tissue sections were consecutively stained with streptavidin/peroxidase complex for 20 min at 37°C before a substrate solution of 3, 3′-diaminobenzidine tetrahydrochloride was added. Sections were then counterstained in hematoxylin. Bright field photographs were obtained with a digital camera connected to the microscope and analyzed with an image analysis system (Leica, Wetzlar, Germany).
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