Eclipse ti e epifluorescence microscope

Callose deposition was detected according to Millet et al. [51 (link)] with minor modifications. Briefly, rice roots amended with 1.2 % biochar or untreated control plants were collected at 7 dpi. Ten root galls from each treatment were collected and fixed in a 3:1 ethanol: acetic acid solution overnight and then dehydrated in ethanol dilutions of 70, 50, and 30 % in sequence. Finally, the root galls were stained with 0.01 % aniline blue solution using vacuum infiltration. Callose deposition of the root galls was examined under UV light using a Nikon Eclipse Ti-E epifluorescence microscope (excitation, 390 nm; emission 460 nm). Quantification of the callose depositions was performed using ImageJ software.

Fluorescence in situ hybridization (Fig 3B) was performed using the QuantiGene^® ViewRNA ISH Cell Assay kit (Affymetrix #QVC0001), according to the protocol provided by Affymetrix. In short, cultured rat hippocampal neurons were fixed in one of the tested fixatives for 10 min on ice and for another 20 min at room temperature. After a washing step, the cells were incubated in the provided detergent solution, followed by probe hybridization for 3 h at 40°C (using standard probes for GAPDH, provided with the kit by the manufacturer). Afterwards, the samples were washed in the provided wash buffer, and signal amplification was done by incubating the samples in pre‐amplifier and amplifier solution for 30 min each at 40°C. Label hybridization was done as well for 30 min at 40°C using Cy5 as dye. After washing in wash buffer and PBS, the samples were embedded in Mowiol and imaged using an inverted Nikon Eclipse Ti‐E epifluorescence microscope.

The cells were seeded into four-well chamber slides (Lab-Tek II CC2 Chamber Slides), grown and fed for 24 h with EC⁺⁺, followed by 24 h feeding with EC^--. After experimental procedures, the cells were fixed with 4% paraformaldehyde (PFA), washed and permeabilized using 0.1% Triton X-100 for 5 min. The cells were blocked with 1% Bovine Serum Albumin (BSA) containing 2% goat serum (blocking solution) at room temperature for 1 h followed by incubation with the primary antibody in the same blocking solution (mouse monoclonal (mAb), OATP-A (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C [38 (link)]. Next day, the cells were washed three times with 0.1% BSA solution in PBS and were then stained with fluorescence-tagged corresponding secondary antibody at room temperature for 2 h followed by three times wash with PBS. Slides were mounted with 4,6-diamidino-2-phenylindole (DAPI) containing Prolong Gold antifade mounting media (Invitrogen). The slides were allowed to dry, protected from light. Slides were observed and images were obtained using a Nikon Eclipse Ti-E Epi-Fluorescence microscope. Mean fluorescence intensity (MFI) was calculated for each color channel while using Nikon industrial microscope software, NIS-elements AR (advanced research) software 4.13.05. The negative control was only incubated with secondary antibody.