Iterative immunostaining combined with expansion microscopy and image processing reveals nanoscopic network organization of nuclear lamina

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Abstract

Iterative indirect immunofluorescence staining (IT-IF) for expansion and structured illumination microscopy improves superresolution imaging of subnuclear nanostructures. The IT-IF increases antibody labeling density resulting in high intensity, while maintaining the signal-to-background ratio. Together with the robust denoising pipeline, IT-IF reveals the fine structural network of nuclear lamins.

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  1. It looks like each iteration involves permeabilization. How necessary is this step in each iteration rather than simply rounds of primary and secondary antibodies with washes in between?

    Also we’ve found detection of actin with fluorescent phalloidin to require improved signal and reduced background, particularly in photosynthetic cells with a great deal of chlorophyll autofluorescence. This requires 1) low concentrations, 2) reduced incubation time, and 3) brighter fluorophore such as Atto compared to Alexa dyes. For your antibodies, you mention low concentrations also result in low signal, so this is suboptimal so I’m still wondering if we could improve phalloidin staining for difficult samples with iteration. I know you don’t find similar improvements in phalloidin staining with iteration but I’m wondering if your phalloidin staining …

  2. It looks like each iteration involves permeabilization. How necessary is this step in each iteration rather than simply rounds of primary and secondary antibodies with washes in between?

    Also we’ve found detection of actin with fluorescent phalloidin to require improved signal and reduced background, particularly in photosynthetic cells with a great deal of chlorophyll autofluorescence. This requires 1) low concentrations, 2) reduced incubation time, and 3) brighter fluorophore such as Atto compared to Alexa dyes. For your antibodies, you mention low concentrations also result in low signal, so this is suboptimal so I’m still wondering if we could improve phalloidin staining for difficult samples with iteration. I know you don’t find similar improvements in phalloidin staining with iteration but I’m wondering if your phalloidin staining …