Axio imager 2 microscope

Representative images were acquired with a Zeiss LSM700 confocal microscope using a Plan-Apochromat 40x/1.4 objective. Worms were immobilized using 1.5% 1-phenoxy-2-propanol (TCI America, Portland, OR) in M9 buffer and mounted on 5% agar slides. 3D reconstructions were done using Zeiss Zen software as maximum intensity projections. A Zeiss Axio Imager 2 microscope equipped with Chroma HQ filters was used to score AMsh number defects. Any animal with more than the wild type AMshL and AMshR glia were scored as having the defect. Each condition represented 3 experiments of at least 50 D1 animals each that were picked at random from the culture plate unless otherwise noted, in accordance with previous literature in C. elegans. Cell numbers were quantified by counting the number of red nuclei labeled by Pf16f9.3::mCherry::H2B and confirmed by referencing the whole cell morphology labeled by Pf16f9.3::GFP.
For tracking of AMsh cell number during larval development, 10 individual L4 worms per genotype were scored under the Zeiss Axio Imager 2 microscope without 1-phenoxy-2-propanol and recovered from the agar slide. They were scored again when they reached the D1 adult stage.

Formalin-fixed paraffin-embeded sections (4 µm) of whole emrbyos were stained with hematoxylin (Sigma, H9627) for 40 s and with eosin (Sigma-Aldrich, HT110116) for 30 s. The tissue sections were mounted with Permount mounting medium (Fisher Scientific, SP15-100). All procedures were conducted by Molecular Pathology Core, University of Florida. Images were generated with a Zeiss Axio Imager 2 microscope (Carl Zeiss, Inc., Thornwood, NY).

Bracts and apical meristem sections were prepared as described above. ROS detection was performed by staining with DAB [67 (link),68 (link)] (Sigma-Aldrich, Shanghai, China) and HPF [69 (link),70 (link)] (Maokang Bio-technology, Shanghai, China). The samples were examined and captured immediately using a ZEISS Axio Imager 2 microscope (Carl Zeiss, Germany).

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).