Immunofluorescence was conducted as previously described [16 ], with minor modifications. Briefly, coverslips containing either cultured rat neurons or astrocytes were washed with × 1 PBS three times and then were fixed for 15 min in 4% paraformaldehyde in PBS (pH 7.4). After aspiration and rinsing with × 1 PBS, samples were blocked in blocking buffer (5% donkey serum, 0.3% Triton X-100, × 1 PBS) for 1 h. Sections were incubated with primary antibodies (1:500 anti-GFAP, 1:500 anti-βIII-Tubulin, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and secondary antibodies (Alexa Fluor 488, 568 or 647, 1:500, Thermo Fisher Scientific) for 2 h at room temperature protected from light. Coverslips were placed on glass slides containing Prolong® Gold Antifade Reagent with Hoechst 33342 (for nucleus staining) and allowed to cure prior to imaging. Coverslips were imaged on an Olympus BX50 confocal microscope (Olympus, Center Valley, PA, USA) and rendered on Imaris software (Bitplane, Concord, MA, USA).
Bx50 confocal microscope
Manufactured by Olympus
Sourced in United States
The BX50 confocal microscope is a specialized piece of laboratory equipment designed for high-resolution imaging and analysis. It utilizes advanced optical and electronic components to capture detailed, three-dimensional images of microscopic samples. The core function of the BX50 is to provide researchers and scientists with a powerful tool for studying the fine structures and interactions within a variety of biological, materials, and other specimens.
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Lab products found in correlation
Anti β 3 tubulin, by Cell Signaling Technology (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Serum free protein blocking solution, by Agilent Technologies (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Anti zo 1 antibody, by Thermo Fisher Scientific (1 mentions)
Anti rhodopsin antibody, by Abcam (1 mentions)
2 protocols using bx50 confocal microscope
1
Immunofluorescence Labeling of Rat Neurons and Astrocytes
2
Immunohistochemical Analysis of Retinal Tissues
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Retina and RPE/choroid flat mounts, and embedded sections, were deparaffinized and processed for antigen-epitope retrieval. Samples were incubated in a streamer autoclave at 120°C for 10 min in antigen retrieval solution (Histofine, Nichirei Biosciences) and then allowed to cool. Sections were then incubated with protein-blocking serum-free solution (Dako). For double staining, the anti-RPE65 and anti-CRALBP antibodies (Calbiochem) were added to the slides and incubated overnight at 4°C. We also stained additional sections with the following antibodies: anti-4 hydroxynonenal antibody (catalog #STA-035, Cell Biolabs), anti-rhodopsin antibody (catalog #ab3267, Abcam), and anti-ZO-1 antibody (catalog #40-2300, Thermo Fisher Scientific). After washing the slides the following day, the secondary antibodies Alexa Fluor-labeled (594 nm) and red goat anti-rabbit IgG (Molecular Probes) were added and incubated at room temperature for 1 hr. Slides were washed, and anti-fade reagent with DAPI (Prolong Gold antifade reagent with DAPI, Molecular Probes) was added. Pictures were taken within 24 hr using a fluorescence microscope (Leica). Confocal imaging was taken by using an Olympus Fluroview unit and an Olympus BX50 confocal microscope (Olympus).
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