Mitotic spindles have tendency to align v the longest axis that the cell, causing a department plane the is perpendicular come the lengthy axis59–61.

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From: development in molecular Biology and Translational Science, 2014

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Ivana PonjavićKruno VukušićIva M. Tolić, in methods in cabinet Biology, 2021

2 Rationale

Mitotic spindle is a very dynamic and dense structure and it is regularly hard to catch the suitable localization of proteins the are crucial for the spindle, particularly with live microscopy, and also that is why we must rely on addressed specimens and also immunofluorescence to examine the architecture of the spindle. Today, we can use ExM as an boosted version the the immunofluorescence protocol (Chozinski et al., 2016) and also get super-resolution pictures of necessary proteins in the mitotic spindle. Tubulin is perhaps one of the most intriguing protein to visualize in the spindle utilizing ExM and the first example of this method was released in a method paper using tubulin immunostaining the PTK1 cell (Chozinski et al., 2016). Tubulin in the mitotic spindles that zebrafish (Freifeld et al., 2017) and also tubulin-GFP in beforehand metaphase-I spindle of mouse oocytes (So et al., 2019) has also been expanded. Except anti-alpha tubulin and tubulin-GFP, kinetochore protein Hec1 (Chozinski et al., 2016), tetrameric kinesin KIF25 (Decarreau et al., 2017), GFP-Spindly and also mCherry-tubulin (Sacristan et al., 2018) have actually been expanded recently too in mammalian systems.

Here, we define the optimized protocols because that ExM of human mitotic spindles immunostained because that tubulin, and also centromere protein Hec1, and also protocols for ExM the mitotic spindles labeled with endogenously tagged microtubule-associated protein PRC1, which forced adaptations of the initial protocol. One of the most beneficial parts of ours ExM protocol is the protocol for tubulin immunostaining which, also when provided without the expansion, offers plenty that information about the design of the spindle.

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However, we illustrate the greater power of ExM protocols provided to research the mitotic spindle structure, when contrasted to immunofluorescence without expansion, i beg your pardon enable much better structural characterization of characterized structures within the mitotic spindle, such as bridging yarn which connect two sister k-fibers (Kajtez et al., 2016). Moreover, applied protocols permit superior resolution that microtubule bundle distribution in various populations the microtubules, and provide a better insight right into the localization of specific proteins within the spindle.