Apotome mode

Spinal cord (L4-L6) samples were obtained, on the 14th or 35th day after induction from the Trpa1^+/+ e Trpa1^−/− PMS- or RR-EAE-induced mice, respectively. To collect the tissues, animals were transcardially perfused with PBS, followed by 4% paraformaldehyde. Spinal cord samples were removed, post-fixed for 24 h, and cryoprotected (4 °C, overnight) in 30% sucrose. Cryosections (40 µm) of the spinal cord were incubated (4 °C, overnight) with the following primary antibodies: Iba1 [1:1000, Wako, catalog #019–19741, rabbit monoclonal, Wako, Osaka, Japan], GFAP [1:500, Z0334, rabbit monoclonal, DAKO, Santa Clara, CA, USA], anti-Olig2 (1:100, MABN50, Millipore, Darmstadt, DE) diluted in PBS and 2.5% normal goat serum (NGS). Sections were then incubated with fluorescent secondary antibodies: polyclonal Alexa Fluor^® 488, and polyclonal Alexa Fluor^® 594 (1:600, Invitrogen, Waltham, MA, USA) (2 h, room temperature), and cover slipped. Analysis of negative controls (nonimmune serum) was simultaneously performed to exclude the presence of non-specific immunofluorescent staining, cross-immunostaining, or fluorescence bleed-through. Tissues were visualized, and digital images were captured using a Zeiss Axio Imager 2 microscope with Z-stacks in the Apotome mode (Carl Zeiss Microscopy GmbH, Jena, Germany). Data are expressed as mean fluorescence intensity (% of basal).

The dimension of the core–shell microfibers
was characterized using a fluorescence microscope with Apotome mode
(Zeiss, Germany). To visualize individual layers, red and green fluorescence
beads were added to the collagen and alginate solutions at a volume
ratio of 0.2%, respectively during the fiber formation. The dimension
of the fibers was analyzed using Image J software (NIH, Bethesda,
USA). Triplicates per group were tested.