Dmire2 inverted microscope

To determine whether alternative control mechanisms (such as induced systemic resistance) might be elicited by B. velezensis 9D-6, we tested its ability to reduce root colonization by P. syringae DC3000, a Gram-negative bacterium whose growth in vitro was not inhibited by B. velezensis 9D-6. P. syringae DC3000 was labeled using a red fluorescent protein (RFP) reporter gene construct based on the plasmid pME6010 [41 (link), 42 (link)], and used to inoculate Arabidopsis thaliana (L.) Heynh seedlings in hydroponic medium (2.5 × 10⁵ CFU/mL), with or without B. velezensis 9D-6 (2.5 × 10⁶ CFU/mL), as previously described [43 ]. The A. thaliana (L.) Heynh seeds were obtained from the Arabidopsis Biological Resource Center (Columbus, OH, U.S.A) and grown as previously described [43 ].
After seven days, A. thaliana roots were removed from the medium, rinsed in ultrapure water to remove loosely bound material, and attachment by labeled P. syringae DC3000 was imaged using a DMIRE2 inverted microscope with confocal laser scanner (Leica Microsystems GmbH, Wetzlar, Germany). Samples were excited using a helium-neon 543/594 nm laser, and emission was detected at 590–630 nm under a 63 x water immersion objective with a numerical aperture of 1.4.

12 μm-thick frozen sections were mounted with Vectashield with DAPI (Vector). Perfused human or murine vessels were identified as UEA-I-labeled or GS-IB₄-labeled lumenal structures and counted using Leica TCS SP2 Acousto-Optical Beam Splitter confocal system equipped with a DMIRE2 inverted microscope (Diode 405 nm, Argon 488 nm, HeNe 594 nm; Leica Microsystems, Germany) at room temperature. Vessel counting was performed by blinded investigators. A 40x/1.25 oil objective or a 20x/0.7 oil objective was used. Microvessel density (MVD) was analyzed with image sets using ImageJ software (NIH) and reported as vessels/mm².