DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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Frequency sweeps were performed from 10 to 0.1Hz (3 reads per decade) with a
deformation of 2mstrain (amount of deformation, has no units), at 37 o C. The elastic
modulus (E’, also known as Young’s modulus) was calculated using the formula E’=
2G’(1+ν), where G’ is the complex storage modulus measured by the rheometer at 1 Hz
and ν is Poisson’s ratio, assumed to be 0.5 for materials that do not vary its volume
upon stretch, according to the literature (Moore et al., 2010; Saha et al., 2008)
II.2.8 - Immunocytochemistry
Immunocytochemistry was performed on cells cultured on 96 well plates, coverslips or
on functionalized hydrogels (15x15 cm and 24x50 cm). The medium was removed,
MSCs were washed once with PBS 1x and fixed with 4% paraformaldehyde for 15
minutes at room temperature (RT). The fixing reagent was removed and cells were
washed with PBS 1x three times. To stain cells using antibodies, cells were further
permeabilized with PBS-Triton 0.1% for 20 minutes and for 5 minutes with PBS-Tween
0.1% and then blocked with 1% BSA in PBS fort 30 minutes. The primary antibody (anti
O4 and anti Nestin mouse antibodies and anti Beta-III-Tubulin and anti GFAP rabbit
antibodies) were diluted in PBS 1%BSA at the appropriated dilution (1:200) and
incubated overnight (ON) at 4 o C in a humidified atmosphere. The secondary antibodies
used to label the primary antibody, Goat anti-rabbit Alexa-568 (1:200) and Donkey
anti-mouse Alexa-488 (1:200) were diluted in PBS with 1% BSA and let incubating for
1h at room temperature (RT). DAPI was used for nuclear staining, the cells were
incubated with 200ng/ml of DAPI for 5 minutes at RT. For the image acquisition the
stained samples were visualized using a Zeiss Axiovert 200M fluorescence microscope
using AxioVision release 4.8 software (Zeiss). The Image J software was used to analyze
the images.
II.2.9 - Statistical analysis
Statistical analysis was performed by repeated measures one-way ANOVA followed by
Tukey’s test using the software GraphPad Prism (*P