Dm6000 light microscope

M. truncatula wild type and mutant plants were grown in pots (4 × 4 × 4.5 cm³) containing aluminium silicate /sand and inoculated with 200 spores of R. irregularis produced by Premier Tech (Québec, Canada). Mycorrhizal colonized roots at the different time points indicated were collected and stained with ink. The grid line intersect method⁷⁷ was used to quantify mycorrhizal colonization, roots were cut into small segments and spread randomly in plastic petri dishes in which a grid with 1 cm × 1 cm squares was affixed to the base. One hundred and twenty intersections for each root sample were counted to measure roots with or without mycorrhizal infection on a Leica DM6000 light microscope.

Paraffin embedded sections were initially placed in xylene for 5 mins, then into 100% ethanol for 5 mins, then rehydrated by washing in water. Endogenous peroxidase block was performed using 0.3% hydrogen peroxide for 15 mins. The slides were then washed with water, and antigen retrieval was performed by incubating overnight in 1 mM EDTA pH 8.0 with 0.1% Tween in a beaker at 65°C, with stirring overnight. The following day, the beaker was cooled with running water until at room temperature. The slides were then washed with PBS for 5 mins, and then were treated with 100 μl of 2x casein block. The slides were then incubated with 2 μg/ml mouse polyclonal antisera to CD31 (clone JC70, Dako, Ely, UK) or a custom antibody raised against the extracellular region of GRIN2D (Eurogentec, Southampton, UK) for 1 hour. The sections were then stained and visualized using the ImmPRESS universal antibody kit and ImmPACT NovaRed chromagen (Vector labs, Burlingame, CA, USA). The sections were counterstained with Mayer's hematoxylin (Sigma), dehydrated and mounted in distyrene–plasticizer–xylene resin (Sigma). All images were acquired using a Leica DM6000 light microscope (Leica, Milton Keynes, UK). The following tissue arrays were used: MA2, MAN2, MC4, MCN4, CD4, CDN4, CDA3 (Superbiochip, Seoul, Korea).

A 10% neutral buffered formalin solution was used to fix the testis for at least one week prior to HE staining. Approximately 3 mm thick testicular tissues were dehydrated, rendered transparent, and boiled in wax before being embedded in paraffin. The sections (3 μm) were dried for 48 h at 60 ° C and dewaxed in water before being dipped in hematoxylin (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China) for 15 min and washed in tap water for 5 min. Subsequently, the sections were dipped in eosin (Sinopharm Chemical Reagent Beijing Co., Ltd.) for 15 s, then washed again for 5 min. After dehydration of the alcohol gradient, the clearance with xylene and placement of a cover slip were performed. Observations were conducted using the LEICA DM6000 light microscope (Leica, Wetzlar, Germany).