Isolated LA were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 µm sections. Masson trichrome staining was used to evaluate interstitial fibrosis. Images were acquired and digitized on a BZ-9000 Biolevo epifluorescence microscope (Keyence, Osaka, Japan) with an attached digital camera. Fibrotic and normal myocardial tissue areas were analyzed at ×400 magnification using the associated software (Keyence). The percentage fibrosis was figured by calculating the ratio of areas of fibrotic to normal myocardial tissue. For lipid content analysis, 4 sections of the frozen myocardium from each animal were air dried. Sections 5 to 7 μm thick were prepared with oil red O 0.3% wt/vol 2-propanol and distilled water for 15 minutes and then washed with 60% 2-propanol. Five random nonoverlapping fields were captured and digitized by using a BZ-9000 Biolevo epifluorescence microscope (Keyence) at×400 magnification. An area of red (lipid) was selected for its color range, and the proportional area of tissue with this range of color was then digitally quantified and expressed as proportion per area. Four images per atrium were analyzed from 8 animals per group to obtain the mean values.
Bz 9000 biolevo epifluorescence microscope
Manufactured by Keyence
Sourced in Japan
The BZ-9000 Biolevo epifluorescence microscope is a laboratory equipment designed for fluorescence microscopy. It provides high-resolution imaging of fluorescently labeled samples. The core function of the BZ-9000 is to capture and display fluorescent signals from biological specimens.
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5 protocols using bz 9000 biolevo epifluorescence microscope
1
Quantifying Myocardial Fibrosis and Lipids
2
Atrial Fibrillation Appendage Cholinergic Innervation
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Human left atrium appendage specimens from 10 patients with AF (5 paroxysmal, and 5 persistent) who underwent left atrium appendage excision during cardiovascular surgery from January 2015 to April 2020 were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5‐µm sections that were labeled with primary antibodies against choline acetyltransferase (Abcam, Cambridge, UK), and the appropriate biotin‐conjugated secondary antibody (ABC reagent; Vector Laboratories, Burlingame, CA, or DAKO EnVision+ System, Peroxidase; DakoCytomation, Glostrup, Denmark). Images were acquired and digitized on a BZ‐9000 Biolevo epifluorescence microscope with an attached digital camera (Keyence).
3
Quantifying Myocardial Interstitial Fibrosis
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Isolated samples of left atrium (LA), left ventricle (LV), and right atrium (RA) were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5‐μm sections. Masson trichrome staining was used to evaluate interstitial fibrosis. Micrographs were digitized using a BZ‐9000 Biolevo Epifluorescence Microscope (Keyence). Areas of fibrosis were analyzed using an imaging software (Keyence). For each atrium and ventricle, 3 images at a magnification of ×400 were analyzed and averaged.
4
Immunohistochemical Analysis of Inflammatory Markers
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Samples of isolated LA were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5‐μm sections. Some sections were incubated with primary anti‐MCP‐1 or anti‐VCAM‐1 overnight at 4°C and then incubated with appropriate biotin‐conjugated secondary antibody (Vector Laboratories). Sections were visualized using a 3,3′‐Diaminobenzidine Substrates kit (Vector Laboratories) as immunostaining‐positive areas and counterstained with hematoxylin. Other sections were incubated with primary anti‐CD68 or anti‐MCP‐1 and anti‐collagen type 1 overnight at 4°C and then incubated with appropriate biotin‐conjugated secondary antibody (Vector Laboratories). These sections were visualized by immunofluorescence with fluorescein and cyanine 3–conjugated streptavidin and mounted with mounting medium using DAPI to visualize nuclei. Micrographs were digitized using a BZ‐9000 Biolevo Epifluorescence Microscope (Keyence). CD68‐positive cells in 3 images at magnification of ×400 were counted and averaged using imaging software (Keyence), as described.
5
Histological Analysis of Myocardial Fibrosis
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Isolated heart tissue was fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5 mm serial sections, mounted, and processed for tissue histology and immunochemical staining. Masson trichrome staining was used to evaluate interstitial fibrosis. Images were acquired with a BZ-9000 Biolevo epifluorescence microscope (Keyence, Osaka, Japan) with an attached digital camera. Fibrotic and normal myocardial tissues were evaluated at 400Â magnification using Keyence software. The ratio of the area of fibrotic to normal myocardium was calculated and fibrosis was reported as a mean percentage of fibrosis in three images of each atrium. Immunohistochemical staining was with an antibody specific for the F4/80 (Abcam, Cambridge, UK) glycoprotein, a macrophage marker, using a commercially available detection system (Dako, Glostrup, Denmark).
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