Cells were grown on glass coverslips and fixed in either 4% PFA or 100% methanol for 15 min at RT or 10 min at −20°C, respectively. For PFA fixed cells, cell membranes were permeabilized by incubation in 1× PBS, 1% BSA, and 0.2% Triton X-100 for 12 min at RT. Thereafter, cells were incubated in 1× PBS and 2% BSA for 30 min at RT to block unspecific binding of antibodies, followed by overnight incubation with primary antibodies at 4°C. After PBS washing steps, cells were incubated in corresponding Alexa Fluor–conjugated secondary antibodies (Invitrogen) for 45 min at RT. Cell nuclei were labeled by DAPI staining or detected through differential interference contrast (DIC) microscopy. Coverslips were mounted in 1× PBS, 90% glycerol, and 2% N-propyl-gallate on glass slides and sealed with nail polish. Images were captured on a fully motorized BX63 upright microscope (Olympus) with a DP72 color, 12.8-megapixel, 4,140 × 3,096–resolution camera, and DIC. For quantification of receptor localization, the mean fluorescence value of GFP-PDGFRα at a region comprising a 5-µm area, starting from the cell surface into the inside of the cell, was set relative to the fluorescence value of the entire cell. For quantification of c-Cbl fluorescence in the primary cilium, values in the cilium region were set relative to the fluorescence values in background areas of the cytosol.
Bx63 upright microscope
Manufactured by Olympus
Sourced in Japan, United States
The Olympus BX63 is an upright microscope designed for advanced microscopic analysis. It features high-quality optics, precision controls, and a durable construction to support various applications in research and industrial settings. The BX63 provides reliable and consistent performance for a wide range of microscopic observations and measurements.
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Lab products found in correlation
Cellsens dimension v1, by Olympus (3 mentions)
Dp80 camera, by Olympus (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Cellsens software, by Olympus (2 mentions)
Permafluor, by Thermo Fisher Scientific (2 mentions)
Uc90 9 megapixel camera, by Olympus (2 mentions)
Cellsens dimension, by Olympus (2 mentions)
Alexa fluor 647, by Abcam (2 mentions)
Af 401 na, by RnD Systems (2 mentions)
Tyramide superboost, by Thermo Fisher Scientific (2 mentions)
Axio imager m2 upright, by Olympus (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Slide scanner microscope, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Las af software, by Leica (1 mentions)
Cellsens dimension software, by Leica (1 mentions)
Sp 5 mp confocal laser scanning microscope, by Leica (1 mentions)
Pe anti mouse cd31, by BioLegend (1 mentions)
20 protocols using bx63 upright microscope
1
Immunofluorescence Staining of Cultured Cells
2
Quantifying CD8+ T Cell Infiltration
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For mouse tissue, images were collected on an Olympus BX63 upright microscope using a 20×/0.75 UApo/340 objective and captured and white-balanced using a DP80 camera (Olympus) in color mode through CellSens Dimension v1.16 (Olympus). For human tissue, images were acquired on a slide-scanner microscope (Leica) using a 20×/0.30 Plan Achromat objective (Zeiss). Snapshots of the slide-scans were taken using Aperio ImageScope (Leica). Images were then processed and analyzed using Fiji ImageJ (http://imagej.net/Fiji/Downloads ). For mice, 8 fields per region (cortex and choroid) were quantified, for a total area of 2.9 mm2. For each human case, four 4 mm2 fields were quantified in each region, for a total of 16 mm2 per region. Several parameters were measured to assess CD8+ T cell accumulation in each region: absolute number of CD8+ T cells, proportion of vessels with CD8+ T cells, proportion of CD8+ T cells that have transmigrated across the vascular lumen. In human cases both parasitized and non-parasitized vessels were examined. If detected, CD8+ monocytes were not included in counts, and were distinguishable from lymphocytes due to their clearly defined kidney-shaped nuclei. Scoring was performed blinded to CM status by two independent observers, with <10% intra-observer variation found.
3
Immunofluorescence Staining Protocol
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Cells were washed in cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4-2H2O and 1.5 mM KH2PO4), fixed for 30 min at 4 °C in 4% paraformaldehyde (PFA, Sigma-Aldrich, #47608) and washed in PBS. Cells were permeabilized in 0.5% Triton-X-100 (Plusone, #17-1315-01) for 10 min and blocked in 5% Bovine Serum Albumin (BSA, Sigma Aldrich #A7906) for 30 min. Primary antibodies were added over night at 4 °C and secondary antibodies for 1 h at room temperature. Cells were treated with DAPI (Invitrogen, Waltham, USA, #C10595) for 5 min to stain nuclei, washed in 1% BSA and mounted in 2% N-propyl-galleate (Sigma-Aldrich, #P-3130) in PBS/glycerine. Images were collected on an Olympus BX63 upright microscope with a 60 ×/1.35 NA oil objective using Olympus CellSens dimension software or (confocal images) on a Leica SP 5 × MP confocal laser scanning microscope with a 40 × or 60 ×/1.3 NA oil objective using Leica LAS AF software. Z-stacks were captured in intervals of 0.2 µm with corresponding Z-scans. Images were processed in ImageJ. No labeling was detectable in the absence of primary antibody at the settings used (data not shown).
4
Murine Brain Immunofluorescence Staining
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Brains were sectioned at 10 µm on a cryostat (Leica), mounted on SuperFrost Plus slides (Thermo Fisher) and fixed with 4% paraformaldehyde for 10 min. Slides were washed with PBS, and non‐specific binding blocked (1% BSA, 0·05% Tween‐20 in PBS) for 1 h, followed by overnight incubation at 4 °C with Alexa Fluor® 647 anti‐mouse CD45·1 (1:100, BioLegend) and PE anti‐mouse CD31 (1:100, BioLegend). Slides were then washed with PBS, incubated with DAPI (0·5 µg/ml) for 30 min, washed again with PBS and covered‐slipped with ProLong Gold (Invitrogen). Images were captured using an Olympus BX63 upright microscope through CellSens Dimension v1·16 (Olympus).
5
Gingival Immune Cell Morphology
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Monocytes and macrophages FACS purified from the gingiva were mounted on Superfrost slides (Thermo Fisher Scientific) using a Cytospin centrifuge (Cytospin 4; Thermo Fisher Scientific). Cells were fixed with ice-cold methanol and stored at room temperature before staining with hematoxylin and acidic eosin (Thermo Fisher Scientific) and mounted with DPX (Thermo Fisher Scientific). Images were captured using an Olympus BX63 upright microscope. Images were then processed and analyzed using Fiji ImageJ.
6
In situ Hybridization of Fgf22 in Mice
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In situ hybridization was performed as described (Schaeren-Wiemers and Gerfin-Moser, 1993 (link); Terauchi et al., 2010 (link)). Digoxigenin-labeled cRNA probes were generated by in vitro transcription using DIG RNA labeling mix (Roche, Basel, Switzerland). The probe for Fgf22 was generated from the cording region of the mouse Fgf22 cDNA. In situ images were taken with a digital camera (Alpha 5100, Sony, Tokyo, Japan) attached to an Olympus BX63 upright microscope (Olympus, Tokyo, Japan) under bright-field optics with 10× objective lenses.
7
Cassiosome Prey Interaction in Microfluidics
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To observe the effects of cassiosomes on potential prey items in relatively confined spaces, discharge assays were conducted within a microfluidic device into which Artemia nauplii (1–2 days old) were introduced along with cassiosomes either isolated from or still within the mucus in FASW. This 6-chambered microfluidic device, with chambers 7-mm wide × 400-μm tall × 3-mm long, modified from a version originally designed as a behavioral microarena for imaging Hydra, was used to immobilize large cassiosomes (~400 μm diameter) or to slow down the movement of smaller cassiosomes (<400 μm) to conduct observations over periods of up to two days. Furthermore, Artemia nauplii (1–3 days old) were introduced into petri dishes (100-ml) containing either isolated cassiosomes, mucus lacking cassiosomes or a FASW control. Video-documentation was conducted using Nikon Stereoscope with a Nikon D7000 Camera and Sharp Shooter3 software or at the Imaging Lab at NMNH, Smithsonian Institution (Washington D.C.) using an Olympus BX63 upright microscope, and viewed with cellSens software. All movies were trimmed using iMovie and are available as Supplementary Movies.
8
Immunofluorescence Microscopy of FFPE Tissue
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Formalin‐fixed paraffin‐embedded (FFPE) tissue sections were cut at a thickness of 5 μm and, after antigen demasking, were incubated overnight at 4°C with the antibodies in PBS with 2% BSA and 10% preimmune serum for blocking of unspecific binding. The next day, the sections were washed and incubated with the respective secondary antibodies. Alexa Fluor® 546 goat anti‐rabbit, A11035 (diluted 1:500 in PBS with 2% BSA), for 30 min at room temperature and counterstained with 2 μg/mL Hoechst‐33258 (Molecular Probes, H1398) to visualize the nuclei and mounted in Permafluor (Thermo Scientific, TA‐030‐FM) for immunofluorescence microscopy. Images were acquired with identical settings using a BX63 upright microscope equipped with an UC90 9‐megapixel camera operated via the cellSens software at 40× magnification (all Olympus). Subsequently the epidermal staining was evaluated in at least four fields of view in sections of three different mice per genotype and age group.
9
Immunohistochemical Analysis of TWF1 in Tumor Tissue
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The tumour tissues were excised from mice, fixed in 4% paraformaldehyde and then embedded in paraffin for routine slide production. The tissue sections (5 µm) were deparaffinized and gradually rehydrated in a descending series of alcohol dilutions. The tissue sections were incubated in 3% H2O2 for 15 min at room temperature and then heated to 95°C in 10 mM sodium citrate buffer (pH 9.0) for 20 min for antigen retrieval. Then the slides were blocked with 10% goat serum (Abcam, Cambridge, UK) at room temperature for 1 h and subsequently incubated with rabbit antihuman TWF1 primary antibody (Cell Signaling Technology®; diluted at 1:200) at 4°C overnight. The slides were washed in 0.01 M phosphate-buffered saline (PBS, pH 7.2–7.4; Solarbio Life Sciences®, Beijing, China) three times. The slides were incubated with horseradish peroxidase-conjugated antirabbit secondary antibody (Cell Signaling Technology®; diluted at 1:500) at room temperature for 1 h. The slides were washed three times in 0.01 M PBS (pH 7.2–7.4; Solarbio Life Sciences®). The slides were then incubated with 0.05% diaminobenzidine regent (Jiyin Technology®, Shanghai, China) to perform the chromogenic reaction. The slides were observed and photographed using a light microscopy (BX63 upright microscope; Olympus Optical Company, Tokyo, Japan).
10
Immunofluorescence Imaging of Cellular Proteins
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Cells were grown on glass coverslips in 6-well culture dishes, fixed in 4% paraformaldehyde for 15 min at room temperature, washed in icecold PBS, and permeabilized for 12 min in permeabilization buffer (0.2 % Triton X-100, 1% BSA in PBS). Unspecific fluorescence was blocked by a 30 min incubation in PBS plus 2% BSA, followed by incubation with primary antibody for 1 h at room temperature, three washes in 2% BSA blocking buffer, and incubation with secondary antibodies diluted in 2% BSA blocking buffer) for 45 min, washing in PBS containing 2% BSA, a 5 min incubation with (4',6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) for nuclear staining, extensive washing in PBS and mounting in N-propyl-gallate mounting media (2% w/v in PBS/glycerin). Fluorescence images were captured on a fully motorized Olympus BX63 upright microscope with an Olympus DP72 color, 12.8-megapixel, 4.140 × 3.096-resolution camera and with a fully motorized and automated Olympus IX83 Inverted microscope with a Hamamatsu ORCA-Flash 4.0 camera (C11440-22CU). The software used was Olympus CellSens dimension, which is able to do deconvolution on captured z stacks, and images were processed for publication using Adobe Photoshop CS6.
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