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a single neuron dye and image

ronron Posts: 4
in Sample Imaging
I want to patch a neuron and fill it with fluorescent dye (Alexa fluor hydrazide 488, 50 uM) in 350 um slice and image it after CLARITY procedure. Can anyone tell me if this is possible to achieve?
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Comments

  • AdamAdam Posts: 31
    Hi,
    Unless the dye binds to anything in the cell, I would think that it would be removed during the clearing.

    Adam
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  • Kristin_EngbergKristin_Engberg Posts: 46
    Hi Ron - it's hard to say. Hydrazides seem to have a primary amine group, giving the dye potential to bind to the hydrogel in CLARITY. According to the Molecular Probes info sheet for Alexa fluor hydrazides, though, they tend to be quenched after PFA fixation, which is involved in CLARITY. I would say it's definitely worth trying - a 350 um slice will not take long to clear and test out.
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  • SuSamSuSam Posts: 6
    Hi,
    I think direct clearing with clearing reagent will be safer to preserve the dye.
    I remember RapiClear RC1.47 should work with slices within 500um, maybe you can try that first.
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  • ronron Posts: 4
    thank you!
    and if it has biotin, did anyone try staining for it?
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