Paraffin sections (5 μm) were stained with H&E and analysed by light microscopy. Cryosections (10 μm) were incubated with rabbit polyclonal anti-human CD8 (Abcam) and mouse monoclonal anti-human CD4 (DakoCytomation) antibodies, or with rabbit polyclonal anti-human IL-17 (H-132, Santa Cruz Biotechnology) and mouse monoclonal EpCAM (Cell Signalling), followed by secondary species-specific Alexa Fluor 488- or Alexa Fluor 547-conjugated antibodies (Invitrogen). Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Sections were examined under an Olympus BX61 fluorescence microscope (Olympus) and images captured with 10× and 20× magnifications using a F-VIEW II camera (Olympus) and AnalySIS software (Soft Imaging System GmbH).
F view 2 camera
Manufactured by Olympus
Sourced in Germany, Japan
The F-View II camera is a laboratory equipment product designed for microscopy imaging. It features a high-resolution camera sensor and is compatible with a range of microscope models. The F-View II provides clear and detailed image capture capabilities for various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Bx61 microscope, by Olympus (4 mentions)
Cell f software, by Olympus (3 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (3 mentions)
Mz 7.5 fluorescence stereomicroscope, by Leica (2 mentions)
Cell f v2, by Olympus (2 mentions)
Microsuite basic edition software, by Olympus (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Uplanfl 100 oil objective, by Olympus (2 mentions)
Bx60 microscope, by Olympus (2 mentions)
Cellsens software, by Olympus (2 mentions)
Ix 61 microscope, by Olympus (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
System microscope bx 60, by Olympus (2 mentions)
Bx61 fluorescence microscope, by Olympus (1 mentions)
Anti human cd8, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab20034, by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
A0452, by Agilent Technologies (1 mentions)
24 protocols using f view 2 camera
1
Immunofluorescence Analysis of CD8, CD4, and IL-17
2
Alizarin Red S Mineralization Staining
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For fixed specimens, mineral in caudal fins was stained for 1 h in a 0.1% alizarin red S (AR-S) and 0.8% potassium hydroxide (KOH; both from Sigma-Aldrich) solution and tissues were cleared in 1% KOH for 24 h. Fins were gradually transferred to glycerol for final clearing, preservation and imaging35 . For live specimens, fish were immersed for 15 min in a 0.01% AR-S solution (pH 7.4)37 (link), anaesthetized in MS222, photographed and returned to their containers. Staining was performed in groups of 5 (in 200 mL of staining solution) or individually (in 100 mL of staining solution), according to the need of tracking the same individuals over time.
All fins were photographed under a MZ 7.5 fluorescence stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled to a F-View II camera driven by the Cell^F v2.7 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). For each fin, bright field and fluorescence images were collected sequentially to assess the regenerated fin and the mineralized area, respectively. Fluorescence micrographs were taken at λex = 530–560 nm and λem = 580 nm with an exposure of 200 ms for fixed specimens and 500 ms for live specimens.
All fins were photographed under a MZ 7.5 fluorescence stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled to a F-View II camera driven by the Cell^F v2.7 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). For each fin, bright field and fluorescence images were collected sequentially to assess the regenerated fin and the mineralized area, respectively. Fluorescence micrographs were taken at λex = 530–560 nm and λem = 580 nm with an exposure of 200 ms for fixed specimens and 500 ms for live specimens.
3
Quantifying Intratumoral T-Cell Subpopulations
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Intratumoral T-cell subpopulations were detected by a combination of primary antibodies: anti-CD3 (Dako, A0452, Host-rabbit), anti-CD8 (Clone YTC182.20, Abcam, Ab60076, Host-rat), and anti-FOXP3 (Clone 236 A/E7, Abcam, Ab20034, Host-mouse) on acetone-fixed breast tumor cryosections as described earlier [27 (link)]. Primary specific secondary antibodies (anti-rabbit Alexafluor 647, A21245; anti-rat Alexa Fluor 488, A11006; anti-mouse Alexa Fluor 555, A31570) were purchased from Life Technologies. DAPI (Invitrogen, D1306) was utilized to stain cell nuclei.
Total tissue slides were scanned on Olympus IX51 microscope equipped with a F-View II camera (both Olympus) and analyzed by the TissueQuest Cell Analysis Software package (version 4.0.1.0137, TissueGnostics GmbH). For automated analysis with TissueQuest, DAPI staining was used as a master marker for cell identification on the basis of nuclei detection. Based on H&E stained reference slides, regions of interest (ROI) were defined to distinguish between tumor and surrounding non-tumor area. All tissues were analyzed with identical parameters for detection of T cells based on nuclear size, mean staining intensity, and background threshold. Cells were visualized in scattergrams, while the cutoff between positive and negative gated cells was validated manually by backward gating on the original image.
Total tissue slides were scanned on Olympus IX51 microscope equipped with a F-View II camera (both Olympus) and analyzed by the TissueQuest Cell Analysis Software package (version 4.0.1.0137, TissueGnostics GmbH). For automated analysis with TissueQuest, DAPI staining was used as a master marker for cell identification on the basis of nuclei detection. Based on H&E stained reference slides, regions of interest (ROI) were defined to distinguish between tumor and surrounding non-tumor area. All tissues were analyzed with identical parameters for detection of T cells based on nuclear size, mean staining intensity, and background threshold. Cells were visualized in scattergrams, while the cutoff between positive and negative gated cells was validated manually by backward gating on the original image.
4
Zebrafish Diabetes Detection Protocol
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To confirm the diabetic condition of the zebrafish Tg(ins:nfsb-mCherry) males after sperm collection and to avoid blood glucose variations related to stress [48 (link)], males were separated 3 days prior to fish sampling into glass tanks (2 L of water) with 2 males of the same treatment per tank. Therefore, on the sperm sampling day, males of each tank could be sacrificed and sperm collected within 3 min, thus avoiding stress-related blood glucose increases that may have promoted biases in the glucose-related transcripts. Moreover, prior to sampling, males were fasted for 24 h to avoid differential blood glucose fluctuations related to food consumption. The observation of pancreatic fluorescence in Tg(ins:nfsb-mCherry) males was immediately performed. In adult fish, the observation of pancreatic fluorescence was impaired due to the high muscular density surrounding the tissues. Therefore, each fish was dissected and fluorescence was observed under a MZ 7.5 fluorescence stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a green light filter (λex = 530–560 nm and λem = 580 nm) coupled to a black and white F-View II camera (Olympus, Hamburg, Germany), which was controlled by Cell^F v2.7 software (Olympus Soft Imaging, Münster, Germany).
5
Formaldehyde-Fixed Cell Microscopy
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For microscopy analysis, cells were fixed in-culture with formaldehyde (3%) at room temperature for 15 min, collected by low-speed centrifugation and re-suspended in PBS. Cells were then photographed with an Olympus F-view II camera mounted on an Olympus 5 BH-2 microscope through a DApo100UV PL 1.30 oil 160/0.17 objective.
6
Microscopic Fiber Quantification
7
Fluorescent Microscopy of Stress Granules
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Cells were prepared for fluorescent microscopy as described previously.53 (link) Briefly, cells were fixed with 4% paraformaldehyde on ice for 15 min, followed by washes with PBS and 5-min permeabilization in cold methanol. After blocking in 5% goat serum/PBS/0.1% Triton X-100 for 1 h at room temperature, coverslips were incubated with primary antibodies diluted in blocking solution for 1 h at room temperature or at 4 °C overnight. Secondary fluorochrome-conjugated antibody was added for 1 h at room temperature in dark place and nuclei were stained with DAPI. Coverslips were mounted on glass slides, on drop of Immumount mounting media (ThermoScientific, Cramlington, UK). Fluorescent and phase contrast images were taken using BX61 microscope, F-View II camera and Cell F software (all Olympus, Tokyo, Japan). Images were prepared using Adobe Photoshop CS3 (San Jose, CA, USA) or Microsoft PowerPoint 2003 (Reading, UK) software. Cells possessing two or more large (mature) SGs (visualized by anti-TIAR or anti-G3BP1 staining) and total number of cells per a view field (× 100 magnification) were counted in 20 or more randomly chosen fields (total ~200–300 cells per coverslip for SH-SY5Y cells and MEFs and ~400 cells for neurons) and mean ratio value was used for statistics.
8
Tendon Explant Viability Assessment
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Cell viability in tendon explants was assessed after 1 day and 7 days of culture by fluorescent microscopy using the fluorescent stains calcein AM (excitation = 495 nm; emission = 515 nm) to stain live cells and Sytox Blue (excitation = 633 nm, 635 nm; emission = 658 nm) to stain nonviable cells (Life Technologies, Carlsbad, CA, USA). At the time of tissue collection on each day, the explants were incubated in the stain for 30 minutes at room temperature. Tissue images were recorded at 4× magnification using an Olympus F view II camera and Micro Suite Basic Edition software (Olympus, Tokyo, Japan). Subjective assessment of viability was performed by six investigators blinded to the treatment and averaged to ensure consistent overall assessment. Each tendon tissue explant was given a score from 0 (0% viability) to 5 (100% viability). The scores from all investigators were averaged to obtain a mean tenocyte subjective viability score for each explant.
9
Microscopic Analysis of S. coelicolor Morphology
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Morphology and viability of S. coelicolor M145 and ΔglnA3 mutant cells grown in a rich complex YEME–TSB (1:1) medium was analyzed in presence of polyamines. Samples were taken from every culture after 72 and168 h of growth, and obtained perpetrates were observed under phase-contrast microscope under 400× enlargement. To detect live and dead cells, SYTO9 and PI (propidium iodide) stains of the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) were used. The SYTO 9 green fluorescent stain labels cells with intact membranes, as well as those with damaged ones. PI penetrates cells with damaged membranes, decreasing SYTO 9 stain fluorescence when both dyes are present. Thus, in the presence of both SYTO9 and PI, cells with intact cell membranes appear fluorescent green whereas cells with damaged membranes appear red. The staining solution was prepared by mixing 0.75 μl of component A and B in 500 μl of water. Stained cells were analyzed by the fluorescence microscopy using Olympus BX60 microscope with an Olympus UPlanFl 100 × oil objective and an Olympus BX-FLA reflected light fluorescence attachment. Images were taken with the F-view II camera (Olympus), using TxRed and eGFP filter sets for detection of the fluorescent markers. The ImageJ was used for image processing. Significant number of images (10) was analyzed in a minimum of three independent culture analyses.
10
Quantifying Spore Viability using Live-Dead Staining
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About 106 spores were plated onto MS agar and sterile coverslips were inserted in a certain angle of 45°. After five to seven days of incubation at 30°C the coverslips were removed and mounted on slides coated with 1% agarose in PBS.
To detect dead spores, SYTO9 and propidium iodide stains of the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) were used. The staining solution was prepared by mixing 1.5μl of component A and B in 1ml of water. Spores were incubated on the agar plate with 20μl of staining solution for 15 minutes, then coverslips were removed and mounted on slides coated with 1% agarose in PBS. Images were taken with an Olympus System Microscope BX60 equipped with a F-view II camera (Olympus), using TxRed and eGFP filtersets for detection of the fluorescent markers. Fiji version v.149b was used for image processing and the Cell Counter Plugin for spore counting. Live-dead percentage was calculated from the analyses of ~ 700–1800 spores from each strain (S3 Table ).
To detect dead spores, SYTO9 and propidium iodide stains of the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) were used. The staining solution was prepared by mixing 1.5μl of component A and B in 1ml of water. Spores were incubated on the agar plate with 20μl of staining solution for 15 minutes, then coverslips were removed and mounted on slides coated with 1% agarose in PBS. Images were taken with an Olympus System Microscope BX60 equipped with a F-view II camera (Olympus), using TxRed and eGFP filtersets for detection of the fluorescent markers. Fiji version v.149b was used for image processing and the Cell Counter Plugin for spore counting. Live-dead percentage was calculated from the analyses of ~ 700–1800 spores from each strain (
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