EYFP fluorescence was measured with a CoolSnap HQ2 CCD camera (Photometrics, Tucson, AZ) using a Nikon Eclipse Ti-U microscope. Excitation was from an X-Cite 120Q light source (EXFO, Mississauga, Ontario), and fluorescence was detected using a Nikon B-2E/C filter set. All images were taken with the same acquisition parameters and analyzed with the ImageJ1.42q software.
B 2e c filter
Manufactured by Nikon
The B-2E/C filter is a photographic filter designed for use with Nikon camera equipment. It is a color correction filter that helps to adjust the white balance and color temperature of the captured images. The filter is made of high-quality glass and is designed to be durable and long-lasting.
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3 protocols using b 2e c filter
1
Measuring EYFP Fluorescence in Microscopy
2
In Vitro Retina Preparation and Intracellular Injection
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The in vitro retina preparation and intracellular injection procedure have been described previously (Dacey and Lee, 1994 ). Eyes were removed from deeply anesthetized animals, and the retina, choroid, and RPE was dissected free of the vitreous and sclera in oxygenated Ames' medium (Sigma‐Aldrich). The retina‐RPE‐choroid was placed flat, vitreal surface upward, in a superfusion chamber mounted on the stage of a light microscope. Autofluorescent granules were visualized with a blue filter block (Nikon B‐2E/C filter, catalog No. 96107; excitation 490 nm; barrier 515 nm). Targeted cells were intracellularly filled with 2–3% Neurobiotin (Vector) and 1–2% pyranine (Molecular Probes) in 1.0 M potassium acetate using high‐impedance (300–450 MΩ) glass micropipettes. After an experiment, retinas were dissected free of the RPE and choroid, fixed for 2 hours in 4% paraformaldehyde, and rinsed overnight in phosphate buffer (0.1 M, pH 7.4).. Retinas were incubated in 0.1% Triton X‐100 (pH 7.4) containing the avidin‐biotin‐HRP complex (Elite kit; Vector) for 8 hours, rinsed in phosphate buffer overnight, and processed for HRP histochemistry with DAB as the chromogen as described above.
3
Intracellular Labeling of Retinal Cells
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