For the neuron staining, we followed the methods of Zhang et al. [18 (link)]. For colocalization, cortical neurons on coverslips were fixed with 4% paraformaldehyde for 10 min after a brief rinse in prewarmed ECS at room temperature, then permeabilized, and blocked through incubating in PBS containing 0.5% Triton X-100 and 5% BSA for 0.5 hr. Then, the neurons were incubated with primary antibodies to SIL1 (rabbit anti-SIL1, Abcam) and synaptophysin (mouse anti-synaptophysin, Abcam) or SD95 (mouse anti-PSD95, Abcam) in PBS containing 0.5% Triton X-100 overnight at 4°C. After rinsing in PBS containing 0.5% Triton X-100 3 times, neurons were incubated with both Alexa 488-conjugated secondary antibody and Alexa 546-conjugated secondary antibody (donkey anti-rabbit or mouse secondary antibody, Abcam) for 1 h at room temperature. After rinsing with PBS three times, neurons were examined under a 60x, 1.4 numerical aperture oil-immersion objective on an Olympus confocal microscope.
Alexa 546 conjugated secondary antibody
Manufactured by Abcam
Alexa 546-conjugated secondary antibody is a laboratory reagent used for fluorescence detection in various biological assays. It is a secondary antibody that is conjugated with the Alexa Fluor 546 fluorescent dye. This dye has an excitation maximum of 556 nm and an emission maximum of 573 nm, making it suitable for detection in the orange-red region of the visible spectrum.
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2 protocols using alexa 546 conjugated secondary antibody
1
Neuronal Colocalization Analysis Protocol
2
Quantifying Surface and Intracellular Protein Localization
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Cortical neurons plated on slides were rinsed three times in ECS, fixed in 4% paraformaldehyde for 5 min, and blocked with 5% BSA in ECS for 10 min. For surface staining, the neurons were incubated in rabbit anti-GFP primary antibody (Abcam, 1:500) for 15 min, rinsed three times in ECS, and incubated with Alexa-546-conjugated secondary antibody (Abcam, 1:1 000) for another 15 min. For intracellular staining, next the neurons were blocked and permeabilized with 0.1% Triton X-100 in PBS containing 5% BSA, then incubated in primary and secondary antibodies for 1 hr each with three times of rinse. Then the neurons were then examined under a 60×, 1.4 numerical aperture oil-immersion objective on an Olympus confocal microscope equipped with FV1000 software. Surface and intracellular staining were analyzed using Metamorph 5.0 software, and the ratio of surface/intracellular signals after stimulation was normalized to the ratio in transfected neurons before stimulation.
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