Eyes were enucleated under anesthesia before euthanasia. Enucleated eyes were incubated in PBS for 10 minutes at RT and then in 4% paraformaldehyde/PBS for 15 minutes on ice. Retinas were removed carefully with Vanna’s scissors and fine forceps, washed with 0.1% Tween 20/Tris-buffered saline (TBS), and blocked with 2% normal goat serum/TBS for 2 hours. The retinas were stained with anti-mouse antibodies CD4-FITC and FoxP3-eFluor 570 (eBioScience) and Lectin-AlexaFluor647 (Life Technologies) overnight and then with DAPI (Invitrogen, Life Technologies) for 15 minutes, followed by mounting with Slowfade mounting media (Life Technologies). Confocal z-stacked images were obtained through an Olympus 7000 (Center Valley, PA, USA). The confocal images were converted to single channel split images before counting CD4-FITC+, FoxP3-eFluor 570+ or CD4-FITC+, FoxP3-eFluor 570- cells using ImageJ (NIH, Bethesda, MD, USA).
Lectin alexafluor647
Manufactured by Thermo Fisher Scientific
Lectin-AlexaFluor647 is a fluorescently labeled lectin designed for use in various biological applications. It consists of a lectin protein conjugated to the AlexaFluor647 fluorescent dye. Lectins are carbohydrate-binding proteins that can be used to detect and analyze specific glycan structures in samples.
Automatically generated - may contain errors
2 protocols using lectin alexafluor647
1
Immunofluorescent Staining of Retinal Cells
2
Retinal Immune Cell Quantification
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Enucleated eyes were incubated in 4% paraformaldehyde in PBS for 30 minutes on ice, and retinas were dissected from the surrounding tissue, washed in Tris-buffered saline, followed by Tris-buffered saline with Triton X-100. The retinas were then blocked with normal goat serum–containing buffer for 2 hours, and then stained overnight with CD4-FITC, Foxp3-efluor570 (both eBioscience), and Lectin-AlexaFluor647 (Life Technologies). Retinas were then washed, stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 minutes, and mounted in Slowfade mounting media (Life Technologies). An Olympus 7000 (Center Valley, PA, USA) was used to acquire images, and ImageJ (ImageJ.nih.gov/ij/">http://ImageJ.nih.gov/ij/ ; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used to convert z-stacked images to dual color images for counting of CD4-FITC/Foxp3-eFluor570 double positive cells.
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