Following anaesthetization, the heart, kidney, and lung were rapidly removed from rats. Tissues were weighed and fixed in 4% aqueous formalin before being snap frozen in liquid nitrogen; then, they were stored at −80 °C. The fixed tissues were embedded in paraffin, sectioned in 3 µm portions with a microtome, and stained with Masson’s trichrome reagent to be able to observe the collagen present in heart tissues. Perivascular and interstitial fibrosis levels were quantified in heart using the Olympus B×41 microscope (400× magnification) (Marques, et al., 2017) [17 (link)]. Collagen levels were expressed as a percentage of the area of the region using the Image-Pro Plus 6.0 (Media Cybemetics, INC., Rockville, MD, USA).
B 41 microscope
Manufactured by Olympus
Sourced in Japan
The Olympus B×41 microscope is a high-quality optical instrument designed for laboratory and research applications. It features a durable construction, advanced optics, and intuitive controls to provide clear, detailed observations. The core function of the B×41 is to magnify and observe samples, enabling detailed analysis and examination.
Automatically generated - may contain errors
Lab products found in correlation
Normal rabbit serum, by Vector Laboratories (1 mentions)
Dp12 camera, by Olympus (1 mentions)
Target retrieval solution s1699, by Agilent Technologies (1 mentions)
Nis elements br, by National Instruments (1 mentions)
C 5050 zoom digital camera, by Olympus (1 mentions)
Cy2 or cy3 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
8 protocols using b 41 microscope
1
Quantification of Cardiac Fibrosis
2
Immunohistochemistry for von Willebrand Factor
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In the immunohistochemical study, the EnVision method was used according to Tokajuk et al. [60 (link)] using antibodies against von Willebrand factor (vWF) (1:2000–2 h incubation at RT, Polyclonal Rabbit Anti-Human (no cat. A 0082); DakoCytomation, Glostrup, Denmark). Antigen retrieval was performed before commencing immunohistochemical staining for vWF using a Target Retrieval Solution (S1699; Dako, Denmark). A negative control was included in which the antibody was replaced by normal rabbit serum (Vector Laboratories, Burlingame, CA, USA) at the respective dilution (no staining), and a positive control was included using rat lung stained for vWF. The obtained results of immunohistochemical staining were evaluated on an Olympus B×41 microscope with an Olympus DP12 camera under 200× magnification. The intensity of the immunohistochemical reaction was measured using a 0 to 256 grey scale level in which completely black pixels were given a value of 256 and white or bright pixels were given a value of 0.
3
Histomorphological Analysis of Mesenteric Arteries
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Third-order mesenteric arteries were fixed in 10% buffered formalin and embedded in paraffin in a routine manner. Four µm thick sections were cut using a Leica 2025 (Leica Biosystems Inc., Buffalo Grove, IL, USA) rotating microtome and stained by hematoxylin and eosin (H+E). Histomorphological analysis of mesenteric arteries slides was performed using an Olympus B×41 microscope (Olympus Corporation, Tokyo, Japan). Digital images were processed using NIS Elements BR software (NIS-Elements BR 3.2 64-bit NIS AR 3.0) which was used to calculate the width of the media. The measurement of the thickness of the middle layer of the mesenteric arteries was made at a uniform 200× magnification. Five sections from each specimen were measured [59 (link)].
4
Muscle Histopathology and Morphometry
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The samples of muscle gastrocnemius were separated and fixed in 10% formalin, embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin and eosin (H&E) for general histopathological analyses, or with hematoxylin and picrofuchsin by van Gieson for detecting collagen fibers [43 ]. The histopathological profiles of each sample were determined by light microscopy observation. Also, digital microphotographs of stained sections were taken at × 400 magnification using a computer-assisted image analyzing system (it consists of an Olympus B×41 microscope and Olympus C-5050 Zoom digital camera, Japan). Then, the muscle fiber diameters and the area occupied by connective tissue in the muscle bundles were measured using ImageJ software.
5
Immunohistochemical Analysis of Testes
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Decapsulated testes were fixed in 4% paraformaldehyde in PBS overnight at 4°C and embedded in paraffin. Sections were mounted on Superfrost plus slides (Fisher Thermo Scientific), rehydrated and subjected to antigen retrieval by microwave heating as described previously [81 (link)]. Sections were blocked in PBS containing 0.1% Triton and 10% donkey serum for 30 min at room temp and then exposed to the primary antibody overnight at 4°C. After washing 3X with PBS, 0.1% TritonX-100 slides were exposed to the secondary antibody for 30–45 min at room temp (biotinylated IgG, 1:200, Jackson ImmunoResearch), washed briefly, exposed to Cy2 or Cy3 conjugated streptavidin (1:1000, Jackson) for 15 min at RT, washed, and cover-slipped using Vectashield containing DAPI (1 μg/ml). Slides were examined using an Olympus B41 microscope and digital images recorded with an Olympus DP71 camera. Images were processed using Photoshop.
6
Computer-Assisted Sperm Motility Analysis
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Sperm cells were concentrated to 5 × 106 cells/mL in Dulbecco's phosphate-buffered saline solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 1.8 mM KH2PO4). A computer-assisted sperm analysis (CASA) system (ISASv1®. Proiser S.L., Valencia, Spain) was used to analyze sperm motility and motion kinematics as described previously [23 (link)]. Briefly, 20 μL of the sample, after appropriate treatment and incubation, was mounted onto a prewarmed Makler chamber (Makler, Haifa, Israel). The sperm samples were examined under 10 × objective magnification in phase-contrast mode using an Olympus B×41 microscope (Olympus Europe GmbH, Hamburg, Germany). After relaying and digitizing the images, the ISAS program was used to analyze the sperms. A minimum of 100 sperms were recorded per field, and at least five fields were obtained for each condition. Among the motion kinematic parameters of sperm, curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), and mean amplitude of head lateral displacement (ALH, μm) were estimated, where sperm with VCL >5 μm/s were considered motile.
7
Immunohistochemical Profiling of Tumor Tissue
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IHC staining of tumor tissue using AEG-1/MTDH-specific rabbit polyclonal antibody (#13860-1-AP), MAP-LC3 (CST-12741), TGF-β1 (CST-3711), E-cadherin (BS-1097) and N-cadherin (BS-2224) antibodies is performed as described previously [39 (link)]. Briefly, human tumor samples are subjected to deparaffinization, rehydration, and antigen retrieval before the staining procedures are performed. The tissue slides are blocked with 2.5% normal horse serum for 10 minutes. Then, tissue slides are incubated with AEG-1/MTDH-specific rabbit polyclonal antibody, MAP-LC3, TGF-β1, E-cadherin and N-cadherin antibody (dilution 1:50) overnight at 4°C. After the tissue slides are washed, they are incubated with anti-mouse IgG HRP and anti-rabbit IgG HRP secondary antibody for 10 minutes. The slides are stained with 3, 3′-diaminobenzidine (DAB) (Vector Laboratories), counterstained with hematoxylin (Vector Laboratories), dehydrated, treated with xylene, and mounted. All slides are examined and representative pictures are taken using an Olympus B × 41 microscope (Olympus America, Melville, NY) [40 (link)].
8
Evaluating Sperm Motility with CASA
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Sperm motility was evaluated by using a computer-assisted sperm analysis (CASA) system (Integrated Sperm Analysis System V1.0; Proiser S.L.; Valencia, Spain). Samples were placed at 37 °C in a water bath for 5 min, and 5 μL of sperm sample was placed onto a slide previously warmed at 37 °C. Samples were then analyzed under a 10× negative phase–contrast objective (Olympus B×41 microscope; Olympus, Tokyo, Japan). Five different fields were analyzed. The following motility descriptors were evaluated: total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, %), straightness (STR, %), oscillation (WOB, %), the amplitude of lateral head displacement (ALH, μm), and frequency of head displacement (BCF, Hz).
CASA settings were: frames per second (25); particle area (> 4 and <75 μm2); connectivity (6); minimum number of images to calculate the ALH (10). The cut-off value for motile spermatozoa was VAP ≥ 10 μm/s, and for progressively motile spermatozoa it was STR ≥ 75%
CASA settings were: frames per second (25); particle area (> 4 and <75 μm2); connectivity (6); minimum number of images to calculate the ALH (10). The cut-off value for motile spermatozoa was VAP ≥ 10 μm/s, and for progressively motile spermatozoa it was STR ≥ 75%
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