Muscle spindle counts were used to test differences among digastric, mylohyoideus, and plantaris muscles.
A spindle was counted as rotating if it had turned by more than 90° in X/Y.
Count spindle numbers were determined following the stereologic method [ 13, 14].
The number of neurons immunoreactive to c-Fos within NVmes, which holds the cell bodies of the spindle afferents, was counted in animals sacrificed 4, 24 and 96 hours after the injection, and also in 5 Unoperated control rats.
It was decided to count spindles only when they appeared from one 5 μm section to the next in the rostrocaudal series of sections.
(K ) The number of astral microtubule at the mother (red bars) and daughter (gray bars) spindle poles were counted in wild-type and Gravin null MEFs, and cells rescued with the murine Gravin T766A mutant that is unable to interact with Plk1 (n = 5 cells ± SEM).
The high frequency of aneuploidy in animals advanced in age was confirmed also by a different method, namely disruption of the metaphase II spindle by monastrol and counting kinetochores on chromosomes stained by DAPI and CREST [5].
On average seven images per rat were analyzed manually (Leica LAS AF version 2.6.0, Leica Microsystems, CMS GmbH, Mannheim, Germany), whereby increased vimentin expression and change in morphology from cobbled stone like cells towards spindle like cells were counted as cells that underwent EMT.
In addition, the length of cytoplasmic microtubules was increased in the tub4-S74E, -S100E and -S360D mutant cells compared to wild type metaphase cells (only wild type cells in metaphase with a nuclear spindle of 1.5-2 µm were counted) or CDC20-depleted cells (Figure 4F and Figure S5).
Muscle spindle diameter and intrafusal fibre counts were obtained from immunohistological sections and were determined at the level of the annulospiral endings (equatorial plane), identified by NF200 staining; 25 muscle spindles/animal were analysed.
