Nie widefield microscope

Cells were grown on poly-L-lysine coated coverslips and fixed with formaldehyde. Primary antibodies included: mouse monoclonal anti-α-tubulin antibody (1:500, Thermo Fisher 62204), mouse monoclonal anti-γ-tubulin (1:1000, Abcam ab11316), rabbit polyclonal anti-α-tubulin (1:5000, Thermo Fisher PA529444), and anti-α-tubulin antibody (Abcam #AB7750). Secondary antibodies included: goat anti-mouse IgG-Texas Red (1:200, Thermo Fisher T-862), goat anti-mouse IgG-Alexa Fluor 546 (1:500, Thermo Fisher A11003), and goat anti-rabbit IgG-Cy5 (1:1000, Thermo Fisher A10523). DNA was stained with Hoechst 33342 or DAPI. For the cold-stable microtubule assays, cells were incubated an ice-water bath for 15 minutes in Leibovitz’s L-15 media without phenol red (Gibco) with 10% FBS. Images were taken using a DeltaVision Core deconvolution microscope equipped with a CoolSnap HQ2 CCD camera using the 60X objective and the 1.5X magnifier setting. Across each cell, at least 40 Z-plane slices were taken 0.2 μm apart. The images were deconvolved and the 20 z-stacks representing the middle of the cell were max projected in z. All other samples were imaged using a Nikon NiE widefield microscope utilizing 40X magnification and a Nikon DS-Qi1Mc camera or a Zeiss Axioimager M1 microscope utilizing 63x and 100x magnifications.

Fluorescent picture acquisition was done on a Nikon A1R or C2 point scanning confocal microscope; bright field images were taken on a Ni-E wide field microscope (Nikon Imaging Center, University of Heidelberg). Images were analyzed and assembled using NIS-Element AR (Nikon), ImageJ/Fiji, Adobe Photoshop and Illustrator software.
For quantification analysis of each marker gene, positive neurons from 3 to 10 DRG sections per mouse (3–4 mice in total) and 3 sections per human DRG (3 human donors in total) were counted manually using Adobe Photoshop software. Averages from all sections were taken and percentages of double positive cells calculated.

Mannitol was used to induce plasmolysis of plants grown for 6 days on 1/2 MS without mannitol. The mannitol concentration at which root cells plasmolysed was determined using epidermal cell without root hairs, in plants expressing the PM marker GFP-LTI6b. Plants were transferred from the growth media to a liquid 1/2 MS solution containing different concentrations of mannitol, as indicated in Figure 3, 1 hr before counting. Plasmolysed and non-plasmolysed cells were counted under a Nikon Ni-E widefield microscope. From each root, the plasmolysis state of 40 cells was assessed. Cells which had root hairs were not included. A cell was considered as plasmolysed if the corners of the cell had detached/curved. Only cells past the maturation zone of the plant were included. The estimated concentration at which 50% of cells would plasmolyse for each genotype was determined by fitting a Boltzmann sigmoid curve in GraphPad Prism 9 to obtain the ‘V50’.