Figure 3

Immunofluorescence co-localization of the MAb Tit1 5 H1.1 target antigen (titin) with respect to actin filaments and some centrosome/centriole proteins. (A) Fibroblasts of the human adult fibroblast cell-line SA-54 were cultured for 3 weeks, fixed with 4% PFA,permeabilized, and double labeled with Alexa Fluor 488 conjugated to Phalloidin for F-actin (green) and with the MAb Tit1 5 H1.1 primary antibody for titin (specific staining detected with Alexa Fluor 594 conjugated to goat anti-mouse IgG secondary antibody (red). (B,C) Mouse embryonal fibroblasts (MEF7) cultured for 3 days, fixed with 4% PFA, permeabilized, and double labeled with Alexa Fluor 488 conjugated to Phalloidin for F-actin (green) and with the MAb Tit1 5 H1.1 primary antibody for titin (specific staining with Alexa Fluor 594, red). (D) Bowes melanoma cells cultured for 2 days, fixed with 4% PFA, permeabilized, and double labeled with MAb Tit1 5 H1.1 for titin (red) and with goat anti-human ninein for ninein (green), and (E) with MAb Tit1 5 H1.1 for titin (red) and with rabbit anti-human γ-tubulin for γ-tubulin (green). (F) Fibroblasts of the human adult fibroblast cell-line SA-54 were cultured for 3 weeks, fixed with 4% PFA, permeabilized, and double labeled with MAb Tit1 5 H1.1 for titin (red) and with rabbit anti-human pericentrin for pericentrin (green). Note the individual staining of the target antigens of all antibodies with centrioles/centrosomes. Arrows show centrosome/centriole staining. Cell nuclei were stained blue with DAPI (obj. 100x).