Eclipse te2000 s epifluorescence microscope

Recombinant His₆-E7*-SAPKQ expressed from Micro-Tom hairy tissue samples located on glass slides was detected by immunofluorescence after softening tissues with 2% driselase in PBS for 40 min at 37°C following fixation with paraformaldehyde 4% in PBS for 1 h. Thereafter, samples were washed in PBS (pH 7.2) three times for 5 min each time with gentle shaking. Then the target was covered with 10% DMSO and 0.5% NP40 in PBS for 1 h at room temperature. Following removal of permeabilization solution, 5% BSA was applied for 1 h at RT and then, samples were dipped with primary rabbit anti-SAP polyclonal antibody diluted 1:300 and incubated at RT in wet box overnight. Then, the samples were washed three times. After a gentle drying, samples were incubated with anti-rabbit polyclonal antibody conjugated with phycoerythrin (sc-3739 goat anti-rabbit IgG-PE, Santa Cruz) diluted 1:1,000, in the dark for 1 h, washed, added with DAPI staining solution, and incubated in the dark at RT for 10 min. Subsequently, slides were washed three times in PBS. Samples were sealed with glycerol:PBS (1:1) for image collection under Nikon Eclipse TE2000-S epifluorescence microscope equipped with a Hg 100 lamp and filter sets appropriate for DAPI and Cy3 fluorescence.

Culture samples were treated using either a 5 μM solution of SYTO® 9 fluorescent stain, Live-Dead® Stain (SYTO® 9 and propridium bromide), or ViaGram™ Red⁺ Bacterial Gram Stain and Viability Kit (SYTOX® Green nucleic acid stain, 4,6-diamidino-2-phenylindole and Texas Red®-X dye–labeled wheat germ agglutinin) in sterile water (Life Technologies). Dark field fluorescence microscopy was performed using a Nikon Eclipse TE2000-S epifluorescence microscope at 20× or 2000× magnification using a high-pressure Hg light source.