Ovarian tissue was collected, and the appearance of the ovary was observed with the naked eye. After the removed ovaries were fixed in 4% paraformaldehyde for at least 48 h, the ovaries were dehydrated, paraffin-embedded, serially sectioned at 5 μm depth, and mounted on microscope slides. The sections were stained by H&E, and then, the histopathology of ovarian tissue was observed by using BX51 microscopy (Olympus, Japan). The follicles were counted only on those containing an oocyte with a clearly visible nucleus. The follicles were categorized as primordial, primary, secondary, and atretic follicles according to the method described previously [44 (link)].
Bx51 microscopy
Manufactured by Olympus
Sourced in Japan, Germany
The Olympus BX51 is a high-performance upright microscope designed for a wide range of research and industrial applications. It features LED illumination, a focusing mechanism, and compatibility with various optical components and accessories.
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Lab products found in correlation
Dp71 ccd camera, by Olympus (2 mentions)
Galactosidase detection kit, by Abcam (2 mentions)
Hematoxylin eosin staining, by Solarbio (2 mentions)
Masson trichrome staining, by Solarbio (2 mentions)
Phalloidin tetramethylrhodamine b isothiocyanate tritc, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamino 2 phenylindole dapi, by Merck Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Dab sa hrp tunel cell apoptosis detection kit, by Wuhan Servicebio Technology (1 mentions)
Dulbecco s phosphate buffered saline, by Merck Group (1 mentions)
Aperio imagescope 12, by Leica (1 mentions)
Cd30 ber h2, by Agilent Technologies (1 mentions)
Bond 3 autostainer, by Leica (1 mentions)
Envision flex, by Agilent Technologies (1 mentions)
Anti cd206, by BioLegend (1 mentions)
Ttf 1, by Agilent Technologies (1 mentions)
Autostainer system, by Agilent Technologies (1 mentions)
Pan cytokeratin, by Agilent Technologies (1 mentions)
Apoptosis detection kit, by Merck Group (1 mentions)
21 protocols using bx51 microscopy
1
Ovarian Histopathology and Follicle Evaluation
2
Immunofluorescence Analysis of A549 Cells
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A549 investigated cells were fixed in 3.8% paraformaldehyde solution containing 0.2% Triton X-100 (Sigma-Aldrich, USA) for 5 min at 37°C and subjected to immunostaining. To evaluate the effect of angiotensin-(1–7) and miRNA transfections in A549 cellular morphology, the actin filaments were stained in a 1% bovine serum albumin (BSA) solution containing 100 μg/ ml of phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) (Sigma-Aldrich, USA) for 1 h. Next, the nuclei of the cells were counterstained in 3.33 ng/ ml 4’,6 diamino-2-phenylindole (DAPI, Sigma-Aldrich, USA) solution for 5 min. After extensive washes with saline phosphate buffer (PBS, 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8 mM Na2HPO4, pH 7.4), the coverslips containing cells were subsequently mounted onto slides and subjected to microscopic immunofluorescence analysis. Images were obtained with an Olympus BX51 microscopy equipped with corresponding filter sets and a DP71 CCD camera (Tokyo, Japan). One hundred randomly selected cells were analyzed in each investigated group. For phase-contrast analyses required in the in vitro scratching assays (wound healing) and in the cellular invasion chamber assays, the same microscopy was used and images were monitored and quantified using the Image J software (http://rsb.info.nih.gov/ij/ ).
3
Cytological Evaluation of IFCC Cells
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The cell sediments were smeared onto a glass slide, stained by Wright’s method, observed with Olympus BX51 microscopy, and photographed by Olympus DP72 image collecting system. Experienced cytopathologists performed cytological evaluation. The smears were classified according to their cytological features, as follows: 1) IFCC positive: Cells were arranged mainly in loose clusters with the occasional presence of floating cells. Nuclear cytoplastic rate was disturbed. Nuclei were usually eccentric, with a thick nuclear membrane, large prominent nucleoli, and coarsely granular chromatin. 2) IFCC negative: Normal cells are present, or cells showonly milder changes of chromatin. 3) Cancer cell cytolysis: Cancer cells lost cell membrane integrity and with karyolysis, pyknosis, or karyorrhexis. Cell debris are dispersing into extracellular space.
4
Immunohistochemical Analysis of Xenograft Tissues
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Immunohistochemistry on TMAs was performed as previously described (Wegiel et al. 2005 (link)). The staining procedure was performed using a semiautomatic staining machine (Ventana ES, Ventana Inc., Tucson, AZ). For immunohistochemical analysis of xenograft mouse organs, tissues or tumors were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. For histology analysis, the sections were stained with hematoxylin-eosin (H&E) and were subjected to analysis using an Olympus BX51 microscopy. Immunostaining of tumor tissues using antibodies was performed as previously described (Wegiel et al. 2008 (link)). The sections were viewed under an Olympus BX51 microscope at magnification of 20× or 40×. The slides were scanned and viewed; microphotographs were taken by using a high resolution scanner (ScanscopeCS, Aperio, Vista, CA). The staining intensity was scored as 0 (negative), 1 (weakly positive or positive), 2 (moderate positive), 3 (strongly or very strongly positive) using an arbitrary semi-quantitative scale.
5
Quantifying Lipid Droplets in LX-2 Cells
6
Nicotiana benthamiana Nuclear Purification
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Agrobacterium-mediated transient expression on Nicotiana benthamiana leaves was conducted by pressure infiltration as described above. Agrobacterium culture of GFP-expressing strain was adjusted to a final optical density at 600 nm (OD600) 0.4 and the strains expressing the various 2b mutants to 0.2. Four days post-infiltration, nuclei were purified from agroinfiltrated leaf patches as described previously33 (link). Thirty leaf discs from each 2b protein constructs were used for purification, nuclei were resuspended in 250 µl nuclei storage buffer. The presence of the purified nuclei of each construct has been verified by visual observation prior to DAPI staining (1 µl of nuclei in 7 µl of DAPI, 1 µg/ml) with an Olympus bx-51 microscopy using U-MNU2 filter, as well as by protein extraction and staining (1:1 with Laemmli buffer).
7
Quantifying Cellular Senescence via Galactosidase
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To quantify senescence, the Galactosidase Detection Kit (Abcam, Cambridge, UK) was used after the end of treatments agreed to the fabricator’s instructions. The number of blue (marker of cellular senescence) and not colored cells were counted in a minimum of three random fields for each sample. Cells were observed in an Olympus BX51 microscopy (Olympus Corporation, Hamburg, Germany).
8
Quantifying Renal Tubular Cell Apoptosis
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Renal tubular cell apoptosis was determined by a DAB (SA-HRP) TUNEL Cell Apoptosis Detection Kit (Servicebio, G1507; Wuhan, China) as instructed by the protocol. TUNEL-positive signals were examined with an Olympus BX51 microscopy (Olympus, Center Valley, PA). Five randomly selected visual fields were checked for the number of apoptotic cells for each sample.
9
Quantifying Vascular and Neuronal Markers in Rat Brains
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Rat brains (n = 6) were fixed with 4 % paraformaldehyde, and embedded in paraffin and cut into a series of 6 μm thick sections. For a morphological analysis of vessels and newborn neurons, samples were rinsed with Dulbecco’s phosphate buffered saline (Sigma, USA) containing 0.01 % Tween-20 and immersed in 3 % H2O2/methanol for 15 min to inhibit endogenous peroxidase activity. Subsequently, brain sections were incubated with 10 % normal goat serum for 20 min at room temperature, and then incubated with primary antibodies against vWF (1:100) and Tuj1 (1:200) at 4 °C for overnight. Following incubation with secondary antibody and ABC kit, sections were colored with DAB kit, and then stained with hematoxylin as a counterstain. Five slides were taken from each brain and each slide was randomly chose 5 non-overlapping fields to observe the expressions of vWF and Tuj1 under BX51 microscopy (Olympus, Japan). The determinant of vascular density was assessed by the number of positive vWF-vessels/mm2 using the Aperio ImageScope 12.3 (Leica), simultaneously vWF positive vascular perimeter and Tuj1 expression were quantified using Image-Pro Plus software in IBZ.
10
Senescence Induction in Cell Cultures
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Cells were seeded in the Lab-Tek II Chamber Slide System of 8-well plates (Nunc) at 3 × 104 cells/well. After 24 h, cells were treated with H2O2 (50, 100, 150, 200 and 400 μM) for 1h. To evaluate senescence, the Galactosidase Detection Kit (Abcam, Cambridge, UK) was used after the end of H2O2 treatment (at 24, 48, or 72 h) according to the fabricator’s instructions. The number of not colored (negatives) and blue (positive, biomarker of cellular senescence) cells were counted in a minimum of three random fields in each sample. Cells were visualized in an Olympus BX51 microscopy.
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