Ten-micrometer cryosections of the common peroneal/tibial nerve and the sural nerve were prepared using a cryostat (Leica, Blenheim, Germany). Immunohistochemical staining with antibodies against CD11b (rat, 1:250, Serotec, MCA711, Puchheim, Germany) for the detection of monocytes/macrophages (further referred to in the text as “macrophages”) and CD 3 (rat, 1:100, Serotec, MCA1477, Puchheim, Germany) for the detection of T cells were performed following standard methods using 0.02% diaminobenzidine (DAB) as chromogen and hemalaun as a counter staining. Anti-rat IgG were used as secondary antibodies. On negative control sections the primary antibody was omitted. Images were acquired using an Axiophot 2 microscope (Zeiss, Oberkochen, Germany) equipped with a CCD camera (Visitron Systems, Tuchheim, Germany). Immunopositive profiles were quantified manually in at least three sections for each mouse and related to the area of the sections. Data were analyzed using SPOT software (software version 5.2, Spot Software BV, Amsterdam, Netherlands) and ImageJ free software version 1.51f (National Institute of Health, Staten Island, NY, USA).
Ccd camera
Manufactured by Visitron
Sourced in Germany
The CCD camera is a type of image sensor commonly used in scientific and industrial applications. It converts light into an electrical signal, allowing for the capture and digitization of visual information. The CCD camera's core function is to provide a reliable and accurate method of image acquisition for various laboratory and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Axiophot 2 microscope, by Zeiss (2 mentions)
Axioplan 2 imaging microscope, by Zeiss (2 mentions)
Scion image software, by Techcomp Instruments (2 mentions)
Mca1477, by Bio-Rad (1 mentions)
Mca711, by Bio-Rad (1 mentions)
Cryostat, by Leica (1 mentions)
Von willebrand factor, by Agilent Technologies (1 mentions)
Tetramethylrhodamine isothiocyanate labeled phalloidin, by Merck Group (1 mentions)
Eclipse te300, by Nikon (1 mentions)
6 protocols using ccd camera
1
Quantifying Macrophages and T Cells in Nerve Tissue
2
Transwell Migration Assay for Melanoma Cells
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The migration assay was based on the migration of melanoma cells seeded in an upper chamber of transwell insert through a membrane with an 8 µm pore size (EMD Millipore). Cells were seeded into 6-well plates at a density of 2×105 cells/well, cultured for 18–24 h and then transfected with 50 nM of each siRNA Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 6–8 h, the medium was replaced with fresh medium.
A total of 24 h after transfection, cells were detached and collected. The upper chamber was seeded with 1×105 cells of OCM1 and OM431 treated with siART3-1, siART3-3, the cells stably expressing shART3-1 and shART3-3 were contained in DMEM with 1% FBS (200 µl). The lower chamber contained 800 µl DMEM and 20% FBS was added to each well of the 24-well plate. Following incubation at 37°C for 48 h, the upper chamber was fixed with 100% methanol and stained with 0.1% crystal violet for 1 h as described previously (14 (link)). Then, the stained chambers were observed and photographing in microscope using a CCD camera (Visitron Systems GmbH, Puchheim, Germany) on a Zeiss Axioplan 2 imaging microscope (Zeiss AG, Oberkochen, Germany). Magnification: ×4.
A total of 24 h after transfection, cells were detached and collected. The upper chamber was seeded with 1×105 cells of OCM1 and OM431 treated with siART3-1, siART3-3, the cells stably expressing shART3-1 and shART3-3 were contained in DMEM with 1% FBS (200 µl). The lower chamber contained 800 µl DMEM and 20% FBS was added to each well of the 24-well plate. Following incubation at 37°C for 48 h, the upper chamber was fixed with 100% methanol and stained with 0.1% crystal violet for 1 h as described previously (14 (link)). Then, the stained chambers were observed and photographing in microscope using a CCD camera (Visitron Systems GmbH, Puchheim, Germany) on a Zeiss Axioplan 2 imaging microscope (Zeiss AG, Oberkochen, Germany). Magnification: ×4.
3
Quantification of Intraepidermal Nerve Fibers
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For IENFD quantification skin specimens were fixed in fresh 4% buffered paraformaldehyde (pH 7.4) for 30 minutes, washed in phosphate buffer, and stored in 10% sucrose with 0.1M phosphate buffer. Skin samples were then embedded in Tissue Tek®, frozen in 2-methylbutane cooled in liquid nitrogen and stored at -80°C before further processing. Forty-μm (for IENFD count) and 10-μm (for CD77 and double stains) cryostat cryostat sections were immunoreacted with antibodies against the pan-axonal marker protein-gene product 9.5 (PGP 9.5; Ultraclone, UK, 1:800) with goat anti-rabbit IgG labelled with cyanine 3.18 fluorescent probe to visualize nerve fibers. Blood vessels were identified by a monoclonal antibody to factor VIII (von Willebrand factor, Dako, USA, 1:200). Three biopsy sections per site were analyzed with a Zeiss Axiophot 2 microscope (Axiophot2, Zeiss, Germany) with a CCD camera (Visitron Systems, Tuchheim, Germany) and SPOT advanced software (Windows Version 4.5, Diagnostic Instruments, Inc, Sterling Heights, USA) on coded slides by an observer blinded to the identity of the specimen. Epidermal nerve fibers were counted following published rules [25 (link)].
4
Actin Cytoskeleton Visualization in Cells
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Cells were seeded on glass coverslips, fixed at a subconfluent state with 3% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton. After blocking with 10% FCS in DMEM, F-actin (filamentous actin) was stained using tetramethylrhodamine isothiocyanate labeled-phalloidin (Sigma-Aldrich, Steinheim, Germany). Cell micrographs were obtained with a CCD camera (Visitron, Munich, Germany) on a Zeiss Axioplan 2 imaging microscope (Zeiss, Oberkochen, Germany).
5
Quantifying Cell Death via PI Labeling
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PI labelling was used as measure of cell death as previously described [21 (link)], following treatment of organotypic slices with glutamate and/or EDAC, by incubation for 30 min with 5 μg/mL PI in aCSF at 37ºC and in a humidified atmosphere of 5%CO2. After incubation, slices were imaged using a Nikon Eclipse TE 300 Inverted fluorescence microscope (Nikon Instruments Inc., Americas) with a × 4 objective and CCD camera (Visitron Systems, Puchheim, Germany). Images were analysed using Scion Image software (Scion Corporation; www.scioncorp.com ), using the "density slice" function to measure the area of PI fluorescence, as a percentage of the total area of each slice.
6
Assessing Organotypic Culture Viability
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The viability of the organotypic culture was assessed by measuring the permeability of the plasma membrane to the normally impermeable fluorescent, DNA-binding dye, propidium iodide (PI—Sigma-Aldrich, USA)107 (link). On the 14th day in vitro (DIV), 5 µM of PI were added to the culture medium and incubated for 2 h at 37 °C with 5% CO2 in a humidified incubator. Cultures were analyzed under an inverted fluorescence microscope (Eclipse TE 300, Nikon, Tokyo, Japan) using a standard rhodamine filter set. Images were captured using a CCD camera (Visitron Systems, Puchheim, Germany) and analyzed using Scion Image software (Maryland, USA). Quantification of the mean intensity of the PI fluorescence was determined densitometrically, considering the total area of hippocampal tissue.
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