HCC tissue array (BC03119b) was purchased and processed from Alena Biotechnology (Xi'an, China) and photographs were taken by upright microscope (Olympus). And the informed written consent from all participants or next of kin was obtained prior to the research. The nude mice were sacrificed at the end of the xenograft experiment after the tumor dissected. The tissues were fixed with 4% paraformaldehyde. Paraffin sections were prepared and the antigen retrieval was applied with EDTA solution (50 mmol/L Tris, 10 mmol/L EDTA, pH = 9.0) after dewaxing treatment. The tissues were incubated with WSB1 antibody (1:200) and c-Myc antibody (1: 200) at 4 °C overnight. DAB chromogenic kit was used to react with the second antibody. Photographs were taken by upright microscope (Olympus). The evaluation standard of staining intensity is as follows: 0 point for no staining, 1 point for yellow, 2 points for brown and yellow, 3 points for yellowish brown. The score criteria for the proportion of positive cells are as follows: 0 point for less than 10%; 1 point for 10%–40%; 2 points for 40%–70%; 3 points for ≥70%. The sum of the two scores is 2 points for 0–70%; 3 points for ≥70%. Adding the two scores of 0–2 is divided into weak expression; 3 to 6 is divided into strong expression.
Upright microscope
Manufactured by Olympus
Sourced in Japan, Canada, China, United States
The Olympus Upright Microscope is a laboratory instrument designed for detailed observation and analysis of specimens. It provides a stable, precise platform for high-magnification viewing of prepared samples. The microscope utilizes an upright optical configuration to offer clear, detailed images of the subject matter.
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Lab products found in correlation
Dp controller software, by Olympus (2 mentions)
Dp30bw, by Olympus (2 mentions)
Bx51wi, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Multiclamp 700a amplifier, by Molecular Devices (2 mentions)
Pc 21, by Narishige (2 mentions)
Inu uk f1, by Tokai Hit (2 mentions)
Transwell chamber, by Corning (2 mentions)
Biocoat matrigel invasion chamber, by Corning (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Safranin o, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Soft image viewer, by Olympus (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Microtome, by Leica camera (1 mentions)
Hematoxylin and eosin, by HiMedia (1 mentions)
Rabbit anti mouse ki67, by Abcam (1 mentions)
Dab substrate, by BD (1 mentions)
Mhs16, by Merck Group (1 mentions)
Mettl3 antibody, by Proteintech (1 mentions)
110 protocols using upright microscope
1
Evaluation of WSB1 and c-Myc Expression
2
Characterization of Decellularized Liver Scaffolds
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To generate liver scaffolds, native livers and decellularized matrix were sliced into square transverse sections (15 mm × 12 mm × 3 mm).The samples used for histology and immunohistochemistry were fixed in 10% buffered formalin for 24 h. Paraffin sections (4 μm thick) were prepared from embedded samples and stained with H&E to visualize to the cellular components and ECM. Nuclear-specific 4,6-diamidino-2-phenylindole (DAPI) staining was performed to assess the degree of nucleus removal. Slides were also stained with Sirius Red (blue/red for proteoglycans/collagens, respectively) according to established protocols. Immunohistochemistry was performed with an antibody against alpha-galactosyltransferase (α-Gal; 1:50; Abcam, Cambridge, MA), swine leukocyte antigen DR (SLA-DRα; 1:50; AbD Serotec, Oxford, UK), and swine leukocyte antigen 2 (SLA2; 1:30; Genetex, San Antonio, TX) to determine the clearance of the Gal epitope, SLA-DRα antigen and SLA2 antigen, respectively. Photomicrographs were taken with an Olympus upright microscope (Olympus, Tokyo, Japan) and an Olympus soft image viewer.
3
Immunohistochemical and Immunofluorescence Analysis
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After heating for 20 minutes at 60°C, liver or ileum paraffin sections were deparaffinized in xylene and rehydrated in decreasing ethanol concentrations, washed in PBS, and blocked in bovine serum albumin for 30 minutes at room temperature. For immunohistochemistry, liver sections were incubated overnight at 4°C with antibodies against TNF-α, interferon-γ, IL-1β, IL-17A, and IL-10. Liver sections were also incubated with biotin-conjugated secondary antibodies at room temperature for 60 minutes and stained with 3, 3′-diaminobenzidine. Images were obtained using an Olympus upright microscope (Olympus Corporation, Japan), and Image J software was used to calculate average optical density.
For immunofluorescence (IF) staining, liver sections were stained with primary anti-F4/80 antibody, anti-Bax antibody, or anti-Caspase-3 antibody at 4°C overnight, incubated with the corresponding secondary antibodies for 60 minutes at room temperature the next day, and underwent a DAPI reaction. Ileum sections were stained with primary anti-ZO-1 antibody or anti-occludin antibody at 4°C overnight and incubated with the corresponding secondary antibodies for 60 minutes at room temperature. Cell nuclei were stained with DAPI. Then it was observed under a fluorescence microscope (Nikon, Japan).
For immunofluorescence (IF) staining, liver sections were stained with primary anti-F4/80 antibody, anti-Bax antibody, or anti-Caspase-3 antibody at 4°C overnight, incubated with the corresponding secondary antibodies for 60 minutes at room temperature the next day, and underwent a DAPI reaction. Ileum sections were stained with primary anti-ZO-1 antibody or anti-occludin antibody at 4°C overnight and incubated with the corresponding secondary antibodies for 60 minutes at room temperature. Cell nuclei were stained with DAPI. Then it was observed under a fluorescence microscope (Nikon, Japan).
4
Hind Paw Tissue Extraction and Acute Slicing
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Peripheral tissue was taken from the plantar surface of the hind paw of Sprague-Dawley rats (21 to 25-day-old) following animal sacrifice. Tissue sections (300–350 µm thickness) consisting of skin layers and the tissue beneath were cut using a tissue chopper (McIlwain Model TC752, Mickle Laboratory Engineering Co., Guilford, Surrey, UK). Acute slices were placed in the recording chamber mounted on the stage of an Olympus upright microscope (Olympus) and maintained in HEPES-based physiological buffer.
5
Dual Whole-Cell Voltage Clamp Technique
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Coverslips with cultured cells were transferred to a recording chamber (RC-27L; Warner Instr. Corp., Hamden, Connecticut, USA) adapted on the stage of an Olympus upright microscope (Olympus, Shinjuku-ku, Tokyo, Japan) with infrared and fluorescence attachments. Cells were visualized using the Nomarski optical infrared attachment equipped with DIC (BX51WI; Olympus) and a DP30BW digital camera with DP Controller software (Olympus).
Two piezoelectric micromanipulators (MX7500 with MC-1000 drive; Siskiyou Inc., Grants Pass, Oregon, USA) were used for precise positioning of the micropipettes. Whole-cell recordings were performed in pairs of Novikoff and HeLaCx43-EGFP cells using a dual whole-cell voltage clamp. The extracellular perfusion solution (extracellular solution; Sigma-Aldridge, St Louis, Missouri, USA) contained (in mM): NaCl, 140; CaCl2, 2.5; MgCl2, 2; HEPES, 10; and KCl, 3 (osmolarity was kept stable at 308 mosmol/l). HEKA amplifiers (EPC-10, three channels, Germany) were used to acquire, store, and analyze the data obtained from pairs of cells.
Junctional current (Ij), measured in cell 2 was divided by voltage (Vj), applied to cell 1 to calculate junctional conductance (gj). Data records were digitized at 5 kHz and filtered at 1 kHz.
Two piezoelectric micromanipulators (MX7500 with MC-1000 drive; Siskiyou Inc., Grants Pass, Oregon, USA) were used for precise positioning of the micropipettes. Whole-cell recordings were performed in pairs of Novikoff and HeLaCx43-EGFP cells using a dual whole-cell voltage clamp. The extracellular perfusion solution (extracellular solution; Sigma-Aldridge, St Louis, Missouri, USA) contained (in mM): NaCl, 140; CaCl2, 2.5; MgCl2, 2; HEPES, 10; and KCl, 3 (osmolarity was kept stable at 308 mosmol/l). HEKA amplifiers (EPC-10, three channels, Germany) were used to acquire, store, and analyze the data obtained from pairs of cells.
Junctional current (Ij), measured in cell 2 was divided by voltage (Vj), applied to cell 1 to calculate junctional conductance (gj). Data records were digitized at 5 kHz and filtered at 1 kHz.
6
Cardiac Tissue Histological Analysis
7
Histopathological Analysis of Cedar Wood Nanoencapsulated Anopheles Larva
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To study the mode of action of cedar wood nanoencapsulated on larva, the treated dead larva of Anopheles was studied through histopathology. The mortal larvae was removed from the treated solution and stored in buffered formalin reagent (pH 7.2). The larva was dehydrated by passing through graded ethanol series and embedded in paraffin wax67 (link). A thin longitudinal section (LS) of the larva stained with Hematoxylin and Eosin (Hi-Media labs) was cut using microtome (Leica, Germany) and mounted on glass slide. The LS of the midgut region were examined under an Upright Olympus microscope. A setup containing tween, span and water served as control was also performed for reference.
8
Evaluating Brain Slice Health Metrics
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Brightfield images were taken on an upright microscope (Olympus) with 4x objective. Cultures’ “health” was evaluated based on three morphological criteria: 1. Blurriness of the culture edge. Blurry edges indicate that the slice has attached well to the polylysine substrate, while distinct edge indicates that the slice has not integrated well with the substrate. Very unhealthy slices become completely detached and float in the culture media. 2. Brightness of the slices. Unhealthy slices appear darker than healthy slices due to accumulation of cellular debris. Brightness was quantified by calculating mean greyscale value of the slice area minus the background in bright field images (Fiji/ImageJ). 3. Integrity and distinctness of neural layers. Healthy slices have well-preserved cytoarchitecture with distinct CA1, CA3 neural layers and dentate gyrus (DG).
9
Histological and Immunohistochemical Analysis of Skin Tissue
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Mice were euthanized after treatment, and approximately 1 cm2 of dorsal skin was excised and fixed in a buffered 4% paraformaldehyde solution. The tissue was then embedded in paraffin, sectioned at a thickness of 5 μm, and subjected to hematoxylin-eosin staining for histological examination. For immunohistochemical (IHC) analysis, sections were incubated with primary antibodies: rabbit anti-mouse Ki67 (1:500; Abcam, United States) and rabbit anti-mouse interleukin (IL)-17 (1:200; Abcam, United States). This was followed by application of a secondary antibody reagent kit (Maxim; Fuzhou, China) and visualization using DAB substrate (BD Pharmingen, United States). After counterstaining with hematoxylin, the sections were dehydrated, cleared, and mounted on glass slides for observation and measurement under an upright microscope (Olympus Corporation, Japan).
10
Histological Analysis of Mouse Liver
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