Sections were analyzed and the images were captured with an Olympus BX-50 photomicroscope attached to a charge-coupled device (CCD) camera (Olympus DP71, 1.5 million pixels, Olympus Corporation, Tokyo, Japan). Sections between the coordinates (bregma-0.24 mm to −3.60 mm for the periventricular nucleus), determined according to the rat brain atlas [19 ], were used for single and double immunohistochemical labeling. We used cross sections taken at five different levels at the same coordinate and at equal distance for each animal in the rostrocaudal plane for cell counting. In dual indirect immunoperoxidase-labeled sections, the ratio of c-Fos- or pSTAT5-expressing neurons to all neuronostatin neurons was calculated for each animal. Counts were performed ‘blindly’ and by two different researchers, and all neuronostatin neurons in the periventricular nucleus were counted. The intra-group mean and standard error of the mean (SEM) of the percentages obtained for each subject were determined. The analysis of variance between the experimental groups was statistically compared using a one-way ANOVA test, and p < 0.05 was considered significant.
Bx50 photomicroscope
Manufactured by Olympus
Sourced in Japan
The BX50 photomicroscope is a high-quality optical microscope designed for laboratory use. It features advanced optics and illumination systems to provide clear, detailed images of specimens. The BX50 is capable of various magnification levels and can be used for a range of microscopy applications. Its core function is to enable detailed observation and analysis of samples.
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11 protocols using bx50 photomicroscope
1
Immunohistochemical Analysis of Neuronostatin Neurons
2
Tissue Histological Processing Protocol
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Tissues (stomach, small intestine, liver, and kidney) obtained immediately after the experimental period were fixed in 10% neutral formalin for 48 h, dehydrated in ascending alcohol series, and embedded in paraffin wax. Sections of 5 μm thickness were cut from the tissues, and routine hematoxylin and eosin staining was performed. Sections were examined histopathologically, using an Olympus BX50 photomicroscope (Olympus, Tokyo, Japan).
3
Histological Evaluation of Rabbit Conjunctiva
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At the end of the 14th postoperative day, the eyes were enucleated, the rabbits were anaesthetized again, and the globes were enucleated together with the conjunctiva, including the bleb, for histologic examination. Single (right) eyes of the rabbits in the control group were also prepared for histologic examination. The operation site of each enucleated eye was dissected as a blockage, which contained the conjunctiva, Tenon’s capsule, and sclera. The surgical area was indicated by the location of the iridectomy. After this procedure, the animals were allowed to live.
The tissue specimens were fixed in 10% formaldehyde and embedded in paraffin before the sections were cut. Serial sections (4 μm thick) were cut, dehydrated, and stained with hematoxylin and eosin stain for light microscopy examination. The specimens were stained with Masson’s trichrome, and microscopic analysis was performed using ×400 objective of a standard light microscope (BX50 photomicroscope; Olympus Corporation, Tokyo, Japan). Using a specific micrometer attached to the microscope, fibroblasts and MNCs in ten randomly determined sections (area of 50 μm2) were counted, and the average numbers of cells in these zones were analyzed. The values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation.
The tissue specimens were fixed in 10% formaldehyde and embedded in paraffin before the sections were cut. Serial sections (4 μm thick) were cut, dehydrated, and stained with hematoxylin and eosin stain for light microscopy examination. The specimens were stained with Masson’s trichrome, and microscopic analysis was performed using ×400 objective of a standard light microscope (BX50 photomicroscope; Olympus Corporation, Tokyo, Japan). Using a specific micrometer attached to the microscope, fibroblasts and MNCs in ten randomly determined sections (area of 50 μm2) were counted, and the average numbers of cells in these zones were analyzed. The values obtained from the cell counts of the ten serial sections were presented as arithmetic means and standard deviation.
4
Microscopic Examination of Plant Cells
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Fresh cells from suspension and callus cultures were examined in filter-sterilized (0.2 μm) distilled water post-autoclaving (FDW) or autoclaved MS medium under 100× oil objective directly or after applying the bacterial stain, Gram’s crystal violet or safranin (0.1–0.5%). An Olympus BX50 photomicroscope equipped with QImaging Micropublisher RTV 5.0 megapixel digital camera (Olympus, Tokyo) and a Leica DM 2000 optical microscope with a DFC-295 digital live camera and Leica Application Suite (LAS) software version 3.8 (Leica Microsystems CMS GmbH, Wetzlar, Germany), were employed for bright-field and phase-contrast microscopy. Live movie files were recorded under bright-field for 20–30 s using the LAS software. The DM2000 microscope allowed generous horizontal scanning and 30–40 µm vertical scanning. Adobe Photoshop (7.0) was used for editing the images, and Microsoft Windows 10.0 Movie Maker for video editing.
5
Microscopic Analysis of Osteogenesis Imperfecta
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Tissue was examined from all 30 OI and 11 normal individuals. Multiple tissue samples were examined since several bones per individual were often obtained (autopsies) and several OI patients had more than one operative procedure. LM assessment initially used a Zeiss photomicroscope with polarization capability and subsequently an Olympus BX50 photomicroscope equipped for polarization studies. Zeiss-examined tissue was photographed using film while tissue examined on the Olympus microscope was recorded by a digital camera system.
6
Tyrosine Hydroxylase Immunohistochemical Staining
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Tyrosine hydroxylase (TH) immunohistochemical staining was developed on free-floating coronal slices. Briefly, sections were treated with 3% H202 and 10% methanol in potassium phosphate buffered saline (KPBS), preincubated with 5% normal goat serum (NGS) and 1% Triton X-100 in KPBS (KPBS-T) for 1 h at room temperature (RT), and later incubated overnight at 4°C with rabbit polyclonal anti-TH (Ref: AB-152, Millipore; 1:1,000) in KPBS/T containing 5% NGS, followed by incubation with a secondary biotinylated goat anti-rabbit IgG antibody (Ref: BA1000, Vector Laboratories, Burlingame, CA; 1:200) in 2.5% NGS KPBS/T for 2 h. Afterward, all sections were processed with the avidin-biotin-peroxidase complex for 1 h using a commercial kit (Ref. PK-6102, Elite ABC kit, Vector Laboratories, Burlingame, CA) and the reaction was shown by using 3,3-diaminobenzidine (DAB).
Images were visualized, captured and analyzed at 4x and 40x magnification by an Olympus BX-50 photomicroscope.
Images were visualized, captured and analyzed at 4x and 40x magnification by an Olympus BX-50 photomicroscope.
7
Immunohistochemical Analysis of EphA4 in Nerve Regeneration
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Sections were incubated in citrate buffer (pH=6.0) at 98°C and then cooled and rinsed for 3×5 min in TRIS tampon (pH=7.6). They were incubated in a 3% H2O2 blocked endogenous peroxidase activity for 5 min at room temperature and rinsed for 3×5 min in TRIS tampon. Blockage of nonspecific binding protein was done by incubating sections with horse serum (Sigma Aldrich, H1138) for 30 min. Incubation with anti-EphA4 primary antibody (Santa Cruz, sc-365503) was done overnight at 4°C. After rinsed for 3×5 min in TRIS tampon, the sections were incubated with donkey anti-mouse IgM secondary antibody (Jackson Immunoresearch, 715-065-140) for 1 h and ABC solution (VECTOR Laboratories, PK6100) for 2 h. The reactions were visualized with DAB and counterstained with Harris haematoxylin.
Results were independently evaluated by two investigators using a light microscope. Fibrosis, inflammation, and degeneration were evaluated on a three-point scale (mild, moderate, and severe). Regenerative changes around the nerve (remyelination and EphA4 receptor expression) were evaluated on a five-point scale (none, poorly, moderate, well, and perfect). Photographs were taken using an Olympus BX 50 photomicroscope.
Results were independently evaluated by two investigators using a light microscope. Fibrosis, inflammation, and degeneration were evaluated on a three-point scale (mild, moderate, and severe). Regenerative changes around the nerve (remyelination and EphA4 receptor expression) were evaluated on a five-point scale (none, poorly, moderate, well, and perfect). Photographs were taken using an Olympus BX 50 photomicroscope.
8
Neuronostatin Neuron Immunoreactivity
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Sections were analysed and photographed with Olympus BX-50 photomicroscope attached to a CCD camera (Olympus DP71, CCD colour camera, 1.5 million pixels, Olympus Corporation, Japan). Sections between the coordinates determined according to the rat brain atlas (bregma -0.24 mm to -3.60 mm for periventricular nucleus) were used for single and double immunohistochemical labelling [24] . Cross-sections were taken at 5 different levels at the same coordinate and an equal distance for each animal. Immunofluorescent staining intensities of neuronostatin neurons co-localised with kainic acid, AMPA and NMDA receptor subunits in the anterior hypothalamic periventricular nucleus were graded by the following scale: '+' was used for a small number of double immunoreactive neuronostatin neurons, '++' for a moderate number of double immunoreactive neuronostatin neurons, and '+++' for a high number of double immunoreactive neuronostatin neurons.
9
Histological Analysis of Rat Organs
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Using cervical dislocation, the control as well as treated rats were sacrificed and the collection of entire tissue specimens were collected from the right lobe of the pancreas, liver & right kidney that were fixed in buffered formalin (10%) and further processed to embed in paraffin wax with the help of routine protocols and microtome cut 5 µm-thick sections. The routine protocol helps to stain the sections with haematoxylin/eosin dye and an Olympus BX50 photomicroscope at 45X helps to examine the stained section. Using light microscopy, the pathological findings of examination were recorded (Attalla et.al., 2010 ).
10
Immunostaining of Growth Factors in Bleb
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Serial slides (4 μm thick) including the bleb site were prepared. The slides were stained with TGF-β, FGF-β, and PDGF kits (Bioss Inc., Woburn, MA, USA) in an automated immunostainer device (BenchMark XT; Ventana Medical Systems, Inc., Tucson, AZ, USA) according to the manufacturer’s instructions. The slides, covered with a special cover substrate, were randomly evaluated under a light microscope (Olympus BX50 photomicroscope). Digital photographs of the tissues were taken at ×40 magnification using the camera on the microscope. Immunostaining intensities of TGF-β, FGF-β, and PDGF were determined semiquantitatively as none (0), weak (1), moderate (2), or intense (3).
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