Fig. 6. Localization of organelles and Ca²⁺-signaling proteins in T cells. T cells were fixed by p-formaldehyde, permeabilized by methanol (except for CD3 staining), and stained for RyR using BODIPY FL-X ryanodine (n=58; B), for Ins(1,4,5)P₃R using anti-Ins(1,4,5)P₃R antiserum and Rhodamine-conjugated secondary antibody (n=46; C), for CD3 using anti-CD3 antibody (OKT3) and a FITC-conjugated secondary antibody (n=17; D), for SERCA using BODIPY-thapsigargin (n=37; E), and for nuclei using Hoechst stain H33258 (n=37; F). Shown are characteristically stained single cells (bar, 5 µm). No or only weak fluorescence was seen in the control cells incubated without primary antibody or a surplus of ryanodine (10-fold) or thapsigargin (100-fold), respectively. All images were acquired from different individual cells. A representative confocal Ca²⁺ image (A; taken from Fig. 2Ab) facilitates comparison of protein and pacemaker signal localization.