Dm 5000b fluorescence microscope

The endogenous levels of NO in shoots and roots were determined by staining with 10 µM 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) fluorescein (Merck, Madrid, Spain) as described previously (Guo et al., 2003 (link)) with slight modifications. Fluorescence was detected by using a Zeiss (Oberkochen, Germany) LSM 780 confocal microscope (with excitation at 488 nm and emission at 500–527 nm range; bandwidth 489–550; gain 1,250) or with a Leica (Wetzlar, Germany) DM 5000B fluorescence microscope with a barrier filter to avoid chlorophyll autofluorescence, using unchanged parameters for every measurement. The specificity of NO-related fluorescence detection was assessed by treatment with 0.5 mM cPTIO or with 0.5 mM salicylic acid (SA) as an NO inducer. The DAF-FM DA fluorescence intensities were analyzed using Adobe Photoshop 7.0 by quantifying green pixels in three to six replicate images taken from independent plants in at least three different pots for every genotype and condition. The number of pixels was always normalized for the cotyledon or root area in each image.
The fluorescence of GFP-tagged NLP7 protein was visualized with a Zeiss LSM 780 confocal microscope (with excitation at 488 nm and emission at 500–527 nm range; bandwidth 489–550; gain 1,250) or a Leica DM 5000B fluorescence microscope in cotyledons or primary root tips, respectively.

Mtb retrieved from both granuloma models were pelleted by centrifugation at 6000 × g for 5 min prior to being inactivated with 1X CellFIX (BD Biosciences) for 20 min at room temperature. Fixed samples were spotted on glass slides, air dried and heat fixed at 70°C for at least 2 h. Acid-fast staining using TB Fluorescent Stain Kit M (BD) was performed in combination with neutral lipid staining dye Nile red (Sigma-Aldrich). Each sample was stained with auramine-O for 20 min, decolorized for 30 s, covered with Nile red (10 μg/ml in ethanol) for 15 min and counterstained with potassium permanganate for 2 min. Samples were gently washed with distilled water between each step. Stained slides were examined using a Leica DM5000 B fluorescence microscope. For quantitative analysis, at least 200 bacteria per sample were counted.

Deparaffinized 3 μm thick sections were pretreated for 20 min with 0.2 M HCl, incubated with 1 M NaSCN for 30 min at 80°C, washed in dH₂O and 2x SCC and digested with 1 mg/ml pepsin (2,500–3,500 U/mg, Sigma Chemical, St. Louis, MO) in 0.14 M NaCl solution, pH2. The biotin labeled full length MCPyV DNA probe was added to the samples at a concentration of 5 ng/μl followed by denaturation of DNA (5 min, 80°C) and hybridization overnight (37°C, humid chamber, Thermobrite, Abbott, IL). Unbound MCPyV DNA probe was stringently washed away in 2x SSC, pH7 at 70°C for 2 min. Bound probe was detected by sequential incubation in a combination of fluorescein isothiocyanate (FITC) biotinylated avidin (AvFITC; 1:500; Vector, Brunswig Chemie, Amsterdam, The Netherlands) and biotin conjugated goat antiavidin (BioGaA; 1:100; Vector). Prior to incubation aspecific binding sites were blocked with Boehringer Blocking reagent. Cell nuclei were counterstained and coverslipped with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; 0.2 μg/ml, Vectashield, Vector Laboratories, CA). Samples were visualized using a DM 5000B fluorescence microscope (Leica, Wetzlar, Germany) coupled to an online digital camera (Leica DC 300 Fx) for independent evaluation of FISH signals by 3 investigators (AzH, DR, LH) according to criteria described earlier (Hafkamp et al., 2008 (link); Haugg et al., 2014 (link)).