The brains of sacrificed mice were removed, fixed in formalin, and embedded in paraffin. For immunofluorescent staining, the primary and secondary antibody was rabbit anti-salmonella (Abcam, Cambridge, MA, USA) and Alexa Fluor 488 chicken anti-rabbit (1:100; Invitrogen, Carlsbad, CA, USA), respectively. The samples were mounted with 4',6-diamidino-2-phenylindole/Antifade (1:200; Invitrogen, Carlsbad, CA, USA). Images of fluorescently immunolabeled sections were acquired using an 80i microscope (Nikon, Tokyo, Japan). Images were captured using Cell-P imaging software (Olympus, Tokyo, Japan) in both the fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate channels.
80i microscope
Manufactured by Nikon
Sourced in Japan, United States, Germany, China
The Nikon 80i microscope is a laboratory equipment designed for high-performance optical microscopy. It features a modular design and advanced optical components to provide clear and detailed imaging across a range of applications. The Nikon 80i is capable of various imaging techniques, including brightfield, darkfield, and polarized light microscopy.
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Lab products found in correlation
Nis elements software, by Nikon (10 mentions)
Dxm1200c camera, by Nikon (6 mentions)
Nis elements f2, by Nikon (4 mentions)
Ds fi3 camera, by Nikon (3 mentions)
Streptavidin hrp conjugate, by Thermo Fisher Scientific (3 mentions)
Permount, by Thermo Fisher Scientific (3 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (3 mentions)
Flour g, by Thermo Fisher Scientific (3 mentions)
Biotinylated goat anti rabbit igg or anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
Image pro plus, by Media Cybernetics (2 mentions)
Hoechst 33342, by Beyotime (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Karyotyping software, by Applied Spectral Imaging (2 mentions)
Prism 8, by GraphPad (2 mentions)
80i camera, by Nikon (2 mentions)
Nis elements ar 3, by Nikon (2 mentions)
Goat anti mouse igg texas red, by Thermo Fisher Scientific (2 mentions)
Proliferating cell nuclear antigen (pcna), by Agilent Technologies (2 mentions)
158 protocols using 80i microscope
1
Immunofluorescent Staining of Mouse Brains
2
Quantifying Adipocyte Lipid Accumulation
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Cells were cultured in 6-well plates and Oil Red O staining was performed as previously described (20 (link)). Briefly, cells were fixed in 4% formalin for 30 min, permeabilized in 60% isopropanol for 20 min, and stained with Oil Red O for 20 min at room temperature. Cells were washed 3 times with distilled water, air dried and counterstained with hematoxylin for 3 min at room temperature. Slides were imaged on a Nikon 80i microscope (magnification, ×10). Counts and lipid droplet areas were analyzed using MetaMorph software (version 6.2; Molecular Devices, LLC). Quantitative analysis of the lipid droplet in adipocytes was measured by spectrophotometry. In brief, Oil Red O staining was dissolved with isopropyl alcohol and the optical density was measured at 510 nm by spectrophotometry.
3
Coelomocyte Imaging: Phase Contrast, DIC, and Polarization
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Coelomocytes were settled onto alcohol-washed glass coverslips that were mounted on perfusion chambers and were viewed on a Nikon (Tokyo, Japan) 80i microscope using either a 40×/0.75 numerical aperture (NA) phase contrast plan objective lens or a 60×/1.4 NA phase contrast planapochromatic objective lens. Digitally enhanced phase contrast images were obtained with a Hitachi (Tokyo, Japan) charge-coupled device (CCD) camera coupled to a Hamamatsu (Hamamatsu City, Japan) Argus-10 real-time digital image processor. Frame-averaged, background-subtracted, and contrast-enhanced images were imported into ImageJ (National Institutes of Health, Bethesda, MD), where additional digital contrast enhancement and frame averaging was performed.
For axis-independent polarization microscopy and differential interference contrast (DIC) imaging, cells in perfusion chambers were viewed on an LC-PolScope (Cambridge Research and Instrumentation, Hopkinton, MA) using a strain-free 60×/1.4 NA DIC planapochromatic objective lens mounted on a Nikon Microphot SA microscope, and images were captured using a Q-Imaging (Surrey, Canada) Retiga camera and processed using Micromanager-based LC PolScope-specific software.
For axis-independent polarization microscopy and differential interference contrast (DIC) imaging, cells in perfusion chambers were viewed on an LC-PolScope (Cambridge Research and Instrumentation, Hopkinton, MA) using a strain-free 60×/1.4 NA DIC planapochromatic objective lens mounted on a Nikon Microphot SA microscope, and images were captured using a Q-Imaging (Surrey, Canada) Retiga camera and processed using Micromanager-based LC PolScope-specific software.
4
Histological Analysis of Mouse Ovaries
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After dehydration, the ovaries of the mice were embedded in paraffin and sectioned. The sections were deparaffinized in xylene three times (for 5 min each), rehydrated in anhydrous ethanol and 95% ethanol twice (for 5 min each), and stained with H&E.
For immunohistochemical analyses, the sections were incubated in 3% H2O2 for 10 min, cooled down to room temperature after boiling in 10 mM sodium citrate buffer (pH 6.0) for 15 min, washed, blocked with 10% donkey serum for 60 min, and incubated with primary antibodies overnight at 4°C. The slides were then washed and incubated with biotin-labeled secondary antibodies. The antibodies were further detected using the VECTASTAIN ABC kit and 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). The slides were then counterstained with H&E and imaged under an 80i microscope equipped with a camera (Nikon, Tokyo, Japan).
For immunohistochemical analyses, the sections were incubated in 3% H2O2 for 10 min, cooled down to room temperature after boiling in 10 mM sodium citrate buffer (pH 6.0) for 15 min, washed, blocked with 10% donkey serum for 60 min, and incubated with primary antibodies overnight at 4°C. The slides were then washed and incubated with biotin-labeled secondary antibodies. The antibodies were further detected using the VECTASTAIN ABC kit and 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA). The slides were then counterstained with H&E and imaged under an 80i microscope equipped with a camera (Nikon, Tokyo, Japan).
5
Histological Analysis of Skin Samples
6
Immunocytochemistry and Super-Resolution Imaging
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For fixation, isolated cortices were treated for 5 min in 2–3% formaldehyde in CIB; coelomocytes were fixed following the methods of Henson et al. (1999) (link). For immunolocalization of RhoA, cortices were fixed using 10% trichloroacetic acid after Yonemura et al. (2004) (link). For blocking buffer, phosphate-buffered saline plus 2% goat serum and 1% bovine serum albumin was used, followed by incubation in appropriate primary and secondary antibodies. Cells were mounted in nonhardening Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA), and conventional epifluorescence microscopic screening of samples was performed on a Nikon (Tokyo, Japan) 80i microscope using a 60×/1.4 numerical aperture (NA) Plan Apochromatic phase contrast objective lens with digital images captured using a Photometrics CoolSnap Cf cooled charge-coupled device (CCD) camera (Roper Scientific, Tucson, AZ). For superresolution 3D SIM (Kner et al., 2009 (link)), we used a DeltaVision OMX 3D-SIM Imaging System (GE Healthcare Bio-Sciences, Pittsburgh, PA) with an Olympus (Tokyo, Japan) 60×/1.42 NA objective lens. Captured images were reconstructed using SoftWoRx software and additional linear adjustments and manipulations made using ImageJ and Photoshop (Adobe, San Jose, CA).
7
Quantification of Lymphocytic Inflammation in Lacrimal Glands
8
Indirect Immunofluorescence Analysis of Pax2 and CD24
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For the indirect immunofluorescence analysis, cells grown on coverslips were fixed with 4% para-formaldehyde for 20 min, followed by permeabilizing with 0.1% Triton X-100 for 30 min (For Pax2 detection). After blocking in 3% bovine serum albumin (BSA), cells were probed with each antibody as indicated. The following antibodies were used: rabbit anti-rat Pax2 polyclonal antibody (1∶400, Santa Cruz), rabbit anti-rat CD24 polyclonal antibody (1∶200, Santa Cruz) and FITC-labeled anti-rabbit secondary antibody (1∶1000, Cell Signaling Technology) [25] (link). The immunofluorescence images were recorded with a fluorescence microscope (Nikon 80i Microscope).
9
Oral and Skin Lesion Cytology and Histopathology
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Fine needle aspiration cytology of oral and skin masses were performed and smears were stained with May Grünwald Giemsa (MGG) and Ziehl-Neelsen (ZN). Samples of all lesions were formalin fixed, paraffin embedded and submitted to histopathological evaluation. Slides were stained with Hematoxylin and Eosin (HE), Ziehl-Neelsen (ZN), Von Kossa and Masson’s Trichrome stain and observed at light microscopy level. Based on the inflammatory response, mineralization, necrosis and fibroplasia evaluated, the histopathological lesions were scored in 3 levels of severity (mild, moderate, and severe) by two different pathologists. Photomicrographs were acquired with a Nikon Digital Sight DS-U1 camera mounted on a Nikon 80-i microscope.
10
Comprehensive Analysis of Wound Healing
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Wound healing was assessed using our previously published assays of re-epithelialization, granulation tissue formation, collagen deposition, and angiogenesis using hematoxylin and eosin, Masson’s Trichrome and CD31 stained cryosections [2 (link),7 (link),33 (link)]. Two wounds per mouse were collected and sectioned from one edge to well past the center. Sections were then selected from the center of the wound by microscopic assessment. Three 10-μm sections judged to be at the actual center of the wound were used for re-epithelialization and granulation tissue thickness measurements. Adjacent three 10-μm sections were used for trichrome staining, CD31 staining, and inflammatory cell staining (described below). For all assays, digital images were obtained using a Nikon Instruments 80i microscope and DS-QI1 digital camera and analyzed using NIS Elements image analysis software (Nikon, Melville, NY, USA).
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