All glomeruli without sclerosis of each sample were analyzed. Digital images of each glomerulus were captured using the AxionCam ICc5 (Zeiss, Germany) digital camera attached to the light microscope on the 40x objective lens. Immunostained cells showing an intense brownish staining and located outside glomerular loop were defined as podocytes. For each case, all immunostained brownish-colored podocytes were quantified in each glomerulus and the area of each glomerulus was measured. The result was expressed in cell density per glomerular area according to Venkatareddy et al. [10 (link)].
Axioncam icc 5
Manufactured by Zeiss
Sourced in Germany
The AxionCam ICc 5 is a digital microscope camera designed for high-quality imaging. It features a 5-megapixel CMOS sensor that captures detailed images. The camera is compatible with a range of microscope systems and can be used for various imaging applications.
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5 protocols using axioncam icc 5
1
Quantifying Podocyte Density in Glomeruli
2
Quantifying Podocyte Markers in Kidney Diseases
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Immunohistochemistry was performed on 2-μm paraffin-embedded fragments using the Novolink nonbiotinylated polymer system (Novolink Polymer Detection System Kit, BL, UK, lot 6067432). The primary antibodies and assay conditions were applied as follows: (1) monoclonal mouse anti-human Wilms’ tumor 1 (WT1), Dako M3561, 1:500 and antigen recovery performed with citrate pH 6.0 buffer; (2) anti-SPON2 polyclonal antibody, Abcam Ab187920, 1:1000 and EDTA pH 9.0 buffer for antigen recovery.
Immunostaining quantification was performed from 40x magnification micrographs of all glomeruli observed in the biopsies of DN, FSGS, MCD and IgAN and 10 glomeruli from the control samples. Mindin expression was described as the percentage of labeled area relative to the total area evaluated, which was evaluated using the AxionCam ICc 5 (Zeiss®) interactive image analyzer system. WT1 expression was quantified using ImageJ 1.53 software, and the result was expressed as the cell density per glomerular area (podocyte/x106 μm3), according to Venkatereddy et al. [15 (link)].
Immunostaining quantification was performed from 40x magnification micrographs of all glomeruli observed in the biopsies of DN, FSGS, MCD and IgAN and 10 glomeruli from the control samples. Mindin expression was described as the percentage of labeled area relative to the total area evaluated, which was evaluated using the AxionCam ICc 5 (Zeiss®) interactive image analyzer system. WT1 expression was quantified using ImageJ 1.53 software, and the result was expressed as the cell density per glomerular area (podocyte/x106 μm3), according to Venkatereddy et al. [15 (link)].
3
Quantitative Histological Analysis of Oral Mucositis
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After deep anesthesia with Ketamine (100 mg/kg) and Xylazine (10 mg/kg), via intraperitoneal injection, followed by euthanasia, the mouth's mucosa was excised.
The schedule of euthanasia, as well as the drug delivery, is outlined inFig. 1 .
The excised mouth's mucosa was dissected to obtain the mucositis-affected areas. The samples were immediately fixed in 10% buffered formalin and submitted to routine histological processing for H&E staining.
The sections were analyzed with a x400 magnification Zeiss microscope (Hallbergmoos) in three fields of each sample by an Axion Cam ICC 5 (Zeiss) computer system digital capture performed by means of a digital camera attached to an optical microscope. The captured images were processed in an automatic morphometry Zen 2012 (Blue Edition) program, in which semi-automatic microscopic field morphometry of leukocytes and vascular sections was performed on the lamina propria underlying the epithelial wound.
The schedule of euthanasia, as well as the drug delivery, is outlined in
The excised mouth's mucosa was dissected to obtain the mucositis-affected areas. The samples were immediately fixed in 10% buffered formalin and submitted to routine histological processing for H&E staining.
The sections were analyzed with a x400 magnification Zeiss microscope (Hallbergmoos) in three fields of each sample by an Axion Cam ICC 5 (Zeiss) computer system digital capture performed by means of a digital camera attached to an optical microscope. The captured images were processed in an automatic morphometry Zen 2012 (Blue Edition) program, in which semi-automatic microscopic field morphometry of leukocytes and vascular sections was performed on the lamina propria underlying the epithelial wound.
4
Assessing Microscopic Colon Damage in DSS-Induced Colitis
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To assess the microscopic damage induced by DSS in mice treated with or without SPI, the colon sections were cut longitudinally, washed with PBS, fixed in 10% buffered formalin for 24 hours, and then processed for paraffin embedding followed by microtome sectioning. Tissue sections (5 μm) were obtained and stained with hematoxylin and eosin. For histopathological analysis, the mucosa, submucosa, muscle layers, and serosa were evaluated. These colon sections were also assessed for the presence of edema, inflammatory infiltrate, and epithelial abnormalities.
Images were captured using a digital video camera (Axion camicC5, Zeiss, Berlin, Germany) coupled to an optical microscope (Nikon Eclipse 50i, Nikon, Melville, NY, USA).
Semi-quantitative analyses of infiltrating edema and epithelial abnormalities were also performed and classified according to the level of tissue involvement as follows: mild (>25%), moderate (25-50%), or severe (>50%). A trained pathologist who was blinded to the treatment performed the histopathological analysis. section and are expressed as nanograms per milliliter per gram of tissue (ng/mL/g of tissue).
Images were captured using a digital video camera (Axion camicC5, Zeiss, Berlin, Germany) coupled to an optical microscope (Nikon Eclipse 50i, Nikon, Melville, NY, USA).
Semi-quantitative analyses of infiltrating edema and epithelial abnormalities were also performed and classified according to the level of tissue involvement as follows: mild (>25%), moderate (25-50%), or severe (>50%). A trained pathologist who was blinded to the treatment performed the histopathological analysis. section and are expressed as nanograms per milliliter per gram of tissue (ng/mL/g of tissue).
5
Comprehensive Renal Biopsy Analysis
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All fields of renal biopsy samples and 40 fields of autopsy kidney fragments, which included glomerular and tubulointerstitial compartments, were analyzed. Immunostained cells showing an intense brownish staining were marked by the observer using the interactive AxionCam ICc 5 (Zeiss, Germany) image analysis system with a 40× objective (final magnification of 1600×). Results were expressed as percentage of marked area compared to total area of the analyzed fields.
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