Ez c1 3.90 freeviewer

The lung adenocarcinoma tissue array slide was blocked with 5% donkey serum albumin in 600 μ1 phosphate-buffered saline/0.03% Triton X-100, (pH 6.0) in a humidified chamber for 1 h at room temperature and then were immunostained with 1:100 anti-TOPK raised in mouse (Santa Cruz Biotechnology) and 1:200 donkey anti-mouse IgG conjugated to Cy2 (Jackson ImmunoResearch Laboratories). Image stacks were captured (×20) at room temperature using laser scanning confocal microscopy (NIKON Clsi Confocal Spectral Imaging System; NIKON Instruments Co., Melville, NY). The intensity of immunofluorescence was scored using EZ-C1 3.90 FreeViewer (NIKON).

All visual observations were made using a Nikon Eclipse TE2000-E inverted confocal microscope (Nikon Corporation, Japan). Bimolecular Fluorescence Complementation (BiFC), which was used to test protein–protein interactions in planta, was monitored three days after agroinfiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens cells (strain GV3101), transformed with combinations of respective plasmids. The plasmids encoded the N-terminal (pSITE-nEYFP-C1) or C-terminal (pSITE-cEYFP-C1) section of YFP linked to the LSUs or other proteins of interest. The 35S::H2B-RFP plasmid (encoding histone 2B fused to red fluorescent protein) was used to visualize the nuclei. A. tumefaciens bacteria were grown for 18 h at 28°C in Yeast Extract Broth (YEB) medium supplemented with 10 μg/ml rifampicin and 50 μg/ml spectinomycin (BioShop, Canada) prior to agroinfiltration. Interactions were tested using a 488 nm laser (Sapphire 488-20 CDRH; Coherent Inc., USA) and a 515/30 filter. For RFP, a 543 nm laser (helium–neon laser; Melles Griot, USA) and a 605/75 filter were used. Image data were analyzed using EZ-C1 3.90 FreeViewer (Nikon Corporation, Japan).

The immunofluorescence procedures have been described by our group previously [18 (link)]. Blocked sections were incubated with a primary antibody mix at +4 °C, overnight, in a wet chamber. The following primary antibodies were used to stain the pancreas: anti-insulin (1:400, Abcam, Cambridge, MA, #ab7842), anti-glucagon (1:400, Abcam, #ab10988), anti-somatostatin (1:400, Abcam, #ab53165). Sections were rinsed in PBS 3 times, 10 min each, and incubated for 2 h at room temperature in a mix of fluorophore-conjugated secondary antibodies in wet chamber protected from light. Appropriate secondary antibodies were used that were conjugated with DyLight 488 (1:400, Jackson ImmunoResearch Laboratories Inc., West Grove, PA), Alexa 555 (1:400, Molecular Probes, Eugene, OR), or Alexa 647 (1:400, Molecular Probes). The same solution was used to dilute primary and secondary antibodies: 1% NDS, 1% BSA, 0.03% Triton X-100. After incubation with secondary antibody, slides were washed in PBS 3 times, 10 min each, and mounted with anti-fading agent Gel/Mount (Biomeda, Foster City, CA). Insulin, somatostatin, and glucagon were labelled by green, blue, and red fluorescent probes, respectively.
Stained slides were viewed using a Nikon C1Si microscope. Images were captured on a Nikon C1Si or C1 Plus confocal microscope, and were analyzed using Nikon software EZ-C1 3.90 Free viewer.