CD8+ T cells and peripheral blood leukocytes were seeded onto glass slides by cytospin centrifugation and fixed in 4% formaldehyde for 10 minutes, followed by incubation with 0.2% Triton-X (VWR, 1.086.031.000) diluted in PBS for 15 minutes. To reduce the immune background, sections were blocked with 10% donkey serum (Sigma, D9663) in PBS for 1 hour, followed by blocking with FAB fragment anti-mouse IgG (Jackson Immunoresearch, 715–007–003, 1:10) for 1 hour. Samples were then probed overnight with mouse anti-Plexin-A4 (Plxna4; R&D Systems, 707201, 1:500) and incubated with Donkey Alexa 568–conjugated secondary antibodies (Molecular Probes, A10037, 1:100) for 45 minutes. Nuclei were counterstained with Hoechst-33342 and mounting of the slides was performed with ProLong Gold mounting medium without DAPI. All steps were performed at room temperature. Microscopy was conducted with an Olympus BX41 microscope and cellSens imaging software.
Donkey alexa 568 conjugated secondary antibodies
Manufactured by Thermo Fisher Scientific
Donkey Alexa 568–conjugated secondary antibodies are fluorescent-labeled antibodies used in immunodetection techniques. These secondary antibodies are generated in donkeys and conjugated to the Alexa Fluor 568 dye, which has an excitation maximum at 578 nm and an emission maximum at 603 nm. They are designed to specifically bind and detect primary antibodies raised in other species, enabling visualization and analysis of target proteins or cellular structures.
Automatically generated - may contain errors
2 protocols using donkey alexa 568 conjugated secondary antibodies
1
Immunofluorescent Staining of Plexin-A4
2
Immunohistochemical analysis of PlexinA4 expression
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