Cy3 red

Anti-rabbit or anti-mouse IgG antibodies-conjugated to Cy2 (green) or Cy3 (red) were purchased from Jackson immunoresearch laboratories (West Grove, PA, USA). Ethidium (Etd⁺) bromide was from GIBCO/BRL (Grand Island, NY, USA), fluoromount-G was from Electron Microscopy Science (Hatfield, PA, USA), Previously described polyclonal anti-Cx40.1, -43, and -Cx45 antibodies were used [11 (link)]. Synthesis and characterization of D4 molecule will be published elsewhere.

A DNA-binding fluorescent dye, SYTOX Green (green) from the set SelectFX (Invitrogen, USA), was used for the coloring of E. faecium L3.
Immunohistochemical detection of protein Bac in Enterococcal clones was carried out with human serum IgA (Sigma) conjugated with horseradish peroxidase (HRP). E.faecium L3-Bac+ was incubated with IgA-HRP conjugate for 30 minutes at 25°C. In order to carry out further fluorescence imaging in a confocal laser microscope, a peroxidase was detected by reaction with goat anti-HRP IgG, conjugated with the fluorochrome Cy3 (red) (Jackson ImmunoResearch, USA). The obtained slides were analyzed by confocal argon laser microscope LSM 710 (Zeiss, Germany) (488 nm) or solid state (561 nm) lasers.

Immunofluorescence was performed according to our previously published work[49 (link)].The following antibodies were used for immunoinfluscent assay: anti-K24 (1:200, Abcam, ab87195), α-tubulin (1:200, Santa Cruz, sc-5286). Secondary antibodies labeled with Alexa 488 (green) and cy3 (red) (1:5000, Jackson ImmunoResearch) were used. Cell nuclei were stained with DAPI (Roche) for 10 minutes at a concentration of 5ug/ml (applied after incubation with secondary antibody). Negative controls stained with secondary antibody alone or Rabbit IgG showed no immunolabelling. The K24 location in keratinocytes and epidermis were detected using an inverted fluorescence microscope (Leica, German). The α-tubulin cytoskeletons of lentivirus-infected keratinocytes were visualized using a confocal laser scanning microscopy (Leica, SP8, Germany).