Eclipse te2000 e inverse confocal laser scanning microscope

Transgenic M. truncatula roots were identified using a stereo microscope (Leica MZ10F, Sohns, Germany). 1–2 cm sections of mycorrhized roots were selected. For the localization of fluorescent reporter proteins, longitudinal root sections were cut by hand with a razor blade. These sections were transferred into a physiological buffer (39 mM Na₂HPO₄x2H₂O, 86 mM NaCl, 22 mM KH₂PO₄; pH 7). GFP (500–520 nm), CFP (460–490 nm) and mCherry (599–621 nm) were detected in the inner root cortex using a hybrid detector and confocal microscopy (Leica TCS SP8 MP, Sohns, Germany). To evaluate, if the detected signals originate from the correct fluorophore, lambda-scans were performed in the range of 498–553 nm (mGFP6), 458–513 nm (CFP), and 575–635 nm (mCherry). Each lambda-scan contained eleven detection steps with 5 (mGFP6 and CFP), and 11 (mCherry) nm bandwidth, respectively. For promoter-GUS studies, an Eclipse TE2000-E inverse confocal laser scanning microscope (Nikon GmbH, Düsseldorf, Germany) and software EZ-C1 (Nikon GmbH, Düsseldorf, Germany) were used.

The transfected N2A cells or HeLa cells were fixed with an acetone/methanol mix (1:2) for 5 min at −20 °C and blocked with 1% bovine serum albumin (BSA) in phosphate-buffered solution (PBS) for 30 min at 37 °C. The primary anti-Cx46 antibody SantaCruz Biotechnology, Inc., Heidelberg, Germany, sc-365394) and anti-Cx26 antibody; (Alomone labs, Jerusalem, Israel ACC-212) were used. The antibodies were diluted, respectively, 1:100 and 1:200 in PBS and added to the cells overnight at 4 °C. The secondary iFluor488™-conjugated anti-rabbit and anti-mouse antibodies (16,608 and 16,528) (AAT Bioquest, Sunnyvale, CA, USA) were diluted by 1:1000 in PBS with 2 μM 4’, 6-diamino-2-phenylndole (DAPI) (Sigma-Aldrich,) for 1 h at 37 °C. The cells were washed with PBS and stored at 4 °C. The cells were imaged with an Eclipse TE2000-E inverse confocal laser scanning microscope (Nikon, Düsseldorf, Germany) with a 60× water immersion objective and the software EZ-C1 (Version 3.80, Nikon, Düsseldorf, Germany).