The immunostaining analysis was performed by using the public domain image analysis software Scion Image for Windows (version beta 4.0.2; developed by Scion Corp), as described elsewhere (Fuentes-Santamaría et al., 2003 (link), 2005b (link), 2007a (link),b (link); Alvarado et al., 2004 (link), 2009a (link)). Immunostained cochlear and CN sections were examined via brightfield illumination using a Nikon Eclipse 80i photomicroscope (Nikon Instruments Europe B.V.). Images were captured using a DXM 1200C digital camera (Nikon Instruments Europe B.V.) that was attached to the microscope. The resultant color images of each field were digitized, and the resultant 8-bit red channel images, containing a grayscale of pixel intensities from 0 (white) to 255 (black), were used for the analysis. Assessors were blind as to whether cochlear or cochlear nucleus tissue was from unexposed or exposed animals.
Dxm1200c
Manufactured by Nikon
Sourced in Japan, United States
The DXM1200C is a digital microscope camera from Nikon. It features a high-resolution CMOS sensor and supports live video capture and image acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (11 mentions)
Act 1c software, by Nikon (7 mentions)
Eclipse 80i microscope, by Nikon (4 mentions)
Eclipse te300, by Nikon (4 mentions)
Eclipse 90i, by Nikon (4 mentions)
Nis elements ar 2, by Nikon (4 mentions)
Nis elements br 3, by Nikon (3 mentions)
Eclipse te2000 u, by Nikon (3 mentions)
Eclipse 50i, by Nikon (3 mentions)
Transwell polycarbonate membrane cell culture insert, by Corning (3 mentions)
Eclipse e600, by Nikon (3 mentions)
Nis ar 2, by Nikon (3 mentions)
Nb100 2284, by Novus Biologicals (3 mentions)
Anti rabbit impress kit, by Vector Laboratories (3 mentions)
3 3 diaminobenzidine staining solution, by Vector Laboratories (3 mentions)
Pertex medium, by Merck Group (3 mentions)
Proteinase k, by Roche (3 mentions)
Eclipse 80i light microscope, by Nikon (2 mentions)
Nis elements ar 4, by Nikon (2 mentions)
Mca341r, by Bio-Rad (2 mentions)
77 protocols using dxm1200c
1
Image Analysis of Immunostained Cochlear Tissues
2
Microscopic Imaging of Yeast Cells
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Microscopic images of yeast cells were captured using a Nikon 80i inverted microscope equipped with a Nikon digital DXM1200C camera and stereo microscope. Images were processed using Adobe Photoshop version 5.5 (Adobe Systems, Corp., San Jose, CA). Fluorescence images were captured by using a Nikon A1R confocal laser scanning microscope.
3
Flow Cytometry Cell Sorting Protocol
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For cell sorting by flow cytometry, whole blood or bone marrow from two uninfected animals were first lysed using NH4Cl, then, FcRs were blocked using cynomolgus macaque serum. Cells were counted and incubated for 30 min with the following antibodies: CD11b (ICRF44), CD45 (D058-1283), CDw125 (REA705), CD123 (7G3), CD3 (REA994), CD20 (LT20), CD8a (BW135/80), CD16 (REA423), CD10 (HI10a), CD14 (TUK4) CD32a (IV.3), and CD66 (TET2). Cell sorting was performed using a FACSAria I flow cytometer (Becton Dickinson). The sorted population was smeared with a cytocentrifuge (Cytospin 2, Thermo Scientific) and then stained using May-Grünwald Giemsa. Images were acquired using a Nikon Eclipse 80i with Dxm 1200C digital camera at 60x magnification. Cells were identified by morphological criteria by a cytologist. Promyelocytes and myelocytes were considered to be pre-neutrophils (PreN), metamyelocytes and band cells immature neutrophils, and segmented neutrophils mature cells.
4
Microalgal Morphological Analysis
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The isolated microalga was examined under a light microscope (LM, Eclipse 80i; Nikon Co., Tokyo, Japan). Images were obtained using a camera (DXM 1200C; Nikon Co., Tokyo, Japan), and the cell size and shape were calculated with an image analyzer (NIS-Elements BR 3.0; Nikon Co., Tokyo, Japan). The cell and shape, chloroplast color and shape, number of pyrenoids, and presence or absence of flagella were recorded.
5
Immunohistochemical Analysis of DRG
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The DRG samples were sectioned to a thickness of 30 µm by a freezing microtome (Thermo Fisher Scientific, Waltham, MA, USA) and maintained at room temperature for 1 hour. Sections were blocked with 5% donkey serum for 30 minutes and incubated with GABAAα2R (AGA-002; Alomone Labs, Jerusalem, Israel) and SP (sc58591; Santa Cruz Biotechnology Inc., Dallas, TX, USA), or GABABR1 (ab90883; Abcam, Cambridge, MA, USA) and GFAP (a marker for SGCs activities, 3670S; Cell Signaling) at 4°C overnight, washed three times with 0.1 M PBS, and then incubated in donkey anti-mouse IgG conjugated Alexa Fluor488 (Thermo Fisher Scientific) and donkey anti-rabbit IgG conjugated Alexa Fluor594 (Thermo Fisher Scientific) for 2 hours under room temperature. Control immunostaining was performed by substituting the primary antibody with normal serum.
SP, GABAAα2R, GFAP, and GABABR1 positive immunoactivity in the bilateral DRGs (C3–C6) was detected from every three randomly selected sections of each DRG by a technician who was blind to the grouping and calculated their average optical intensity using an optical imaging analysis system (DXM1200c; Nikon Corporation, Tokyo, Japan). Some representative section photos were taken using a laser scanning confocal microscope (FV1000; Olympus Corporation, Tokyo, Japan). Digital images were then processed with Adobe Photoshop CS2 (Adobe Systems, San Jose, CA, USA).
SP, GABAAα2R, GFAP, and GABABR1 positive immunoactivity in the bilateral DRGs (C3–C6) was detected from every three randomly selected sections of each DRG by a technician who was blind to the grouping and calculated their average optical intensity using an optical imaging analysis system (DXM1200c; Nikon Corporation, Tokyo, Japan). Some representative section photos were taken using a laser scanning confocal microscope (FV1000; Olympus Corporation, Tokyo, Japan). Digital images were then processed with Adobe Photoshop CS2 (Adobe Systems, San Jose, CA, USA).
6
Cytoskeleton Collapse Assays in U87MG Cells
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Cytoskeleton collapse assays using U87MG cells were performed as previously described [11 (link), 18 (link)] using HEK293 cell-derived conditioned medium containing recombinant sema3B or control conditioned medium from cells containing empty expression vectors. To stabilize pH, HEPES buffer (10 mM, pH 7.2) was added. Cells were photographed after a 30 min incubation in a humidified incubator at 37 °C using a phase-contrast inverted microscope (Eclipse TE2000-U; Nikon, Tokyo, Japan). Equipped with a digital photomicrographic camera (DXM1200C; Nikon) at 20x magnification. The images were acquired with ACT-1C software (Nikon).
7
Quantifying Plexin-A2 Ligand Binding
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U87MG KO clone 54.3 cells infected with a lentiviral vector directing expression of plexin-A2 (PlexA2) or with a variant containing a point mutation in the plexin-A2 semaphorin binding site (PlexA2/A396E). The cells were washed with binding buffer (Hank’s-Buffered Salt Solution with 0.5% BSA, 0.1% NaN3, 5 mM CaCl2, and 1 mM MgCl2 and 20 mM HEPES, pH-7.0) for 15 min and incubated for 45 min at 4 °C with 20-fold concentrated sema6A-Fc conditioned medium containing a 1/5000 fold diluted goat anti-human IgG antibody conjugated with alkaline phosphates (AP) (Jackson ImmunoResearch Laboratories). After the removal of unbound ligand, cells were fixed with 4% PFA for 20 min, and rinsed with 20 mM HEPES pH-7.0, 150 mM NaCl. To destroy intrinsic AP activity, cells were heat inactivated in 65 °C for 30 min. Finally, cells were incubated with 5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium chloride (BCIP/NBT) liquid substrate (Sigma-Aldrich (B1911)). After an hour cells were photographed with a phase-contrast inverted microscope (Eclipse TE2000-U; Nikon, Tokyo, Japan). Equipped with a digital photomicrographic camera (DXM1200C; Nikon) at 10x magnification. The images were acquired with ACT-1C software (Nikon). The mean area of stained cells relative to the mean area of total cells per microscopic field was obtained from 38 microscopic fields using Image-pro premier software.
8
Microscopic Film Thickness Measurement
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To measure the thickness of the films, we adapted the Swiss Roll technique [21 (link)], originally developed to prepare tissues for paraffin or methacrylate embedding for histological analyses followed by optical microscopy (Nikon Eclipse 50i; Nikon, Tokyo, Japan). Films (n = 3) were observed using a light microscope with camera (Nikon digital camera, DXM1200C; Nikon, Tokyo, Japan). Film thickness was estimated processing the pictures with the Axiovision software Rel 4.8 (Carl Zeiss Microscopy GmbH, Germany).
9
Quantitative Immunohistochemical Assessment
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All immunohistochemical sections were assessed blindly and independently by two observers (TR and MA), followed by a joint review for resolution of any differences. The expression of proteins was determined as the mean percentage of positive mucosa epithelial cells, manually counted, with the aid of an ocular grid, in ten non-overlapping, random fields (total magnification, ×400) for each case (labeling index, LI; % labeled cells). Immunopositively stained endothelial and lamina propria cells were excluded from the cell counts. Expression of proteins included in this study was examined in adjacent (semi-serial) sections of each sample. Microphotographs were obtained using a Nikon DXM 1200C digital camera mounted on a Nikon Eclipse 80i microscope and ACT-1C software (Nikon Instruments Inc., Melville, NY, USA).
10
Seminiferous Tubule Morphological Analysis
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Morphological changes of seminiferous tubules were analyzed followed the protocols described by Roboon et al. (2017) 10 . Briefly, two sections per animal were used. Each hematoxylin-eosin stained section was evaluated under a light microscope (Nikon eclipse 08i; Nikon, Bangkok, Thailand, Co., Ltd.) and a picture taken using a computerised image capture system (Nikon digital camera DXM1200c, Nikon, Bangkok, Thailand, Co., Ltd.). The data were shown as percentage of each morphological type of in seminiferous tubule per total number of seminiferous tubules in each section.
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