Immunocytochemistry is an established method to identify the localization of cellular proteins. The aim of the present study was to investigate the effect of different fixation treatments on immunocytochemical labelling of ion channels in enzymatically isolated rat ventricular myocytes from humanely killed rats. Two different fixation protocols were employed. In the first fixation protocol, cells were fixed in paraformaldehyde (PFA, 4%) for 30 min at room temperature and incubated with 0.1% Triton X-100 in PBS (which permeabilizes surface membranes as well as those of intracellular organelles). With the second fixation protocol, fixation and permeabilization was performed by incubating cells with ice-cold 100% methanol (MeOH) for 5 min at -20°C. After PFA or MeOH fixation all cells were subjected to the same protocol. They were washed in PBS and blocked with 10% donkey serum in PBS. Following 14-22 h incubation with primary antibodies at 4°C, cells were washed in PBS, incubated with secondary antibodies conjugated to FITC and visualized using laser scanning confocal microscopy. With these two fixation protocols, labelling of various ion channels was investigated: K_v1.5 (responsible for I_Kur, Fig. 1), K_v4.2 (responsible for I_to), K_v7.1 (K_vLQT1, responsible for I_Ks), K_v11.1 (ERG, responsible for I_Kr), K_ir2.2 (responsible for I_K1), K_ir6.2 (responsible for I_KATP), Ca_v1.2 (α_1C, responsible for I_CaL), Ca_v1.3 (α_1D, responsible for I_CaL), Ca_vα₁ (α_1C and α_1D) and Ca_v3.1 (α_1G, responsible for I_CaT). With PFA fixation, there was a distinct and strong striated pattern of labelling (presumably corresponding to t-tubules) of K_v4.2, K_ir6.2 and Ca_vα₁ and a weak striated pattern of labelling of K_v1.5 (Fig. 1), but no labelling of other ion channels. With MeOH fixation, there was a distinct and strong striated pattern of labelling of Ca_v1.2, Ca_v1.3 and Ca_vα₁, a weak striated pattern of labelling of K_v1.5 and labelling at intercalated discs in the case of K_v1.5 (Fig. 1), Ca_vα₁ and Ca_v3.1, but no labelling of other ion channels. Results are summarised in Table 1. This study clearly demonstrates the importance of different fixation protocols on the labelling of ion channel proteins in myocytes. It is concluded that, to visualise ion channels in the heart using immunolabelling, the results obtained from both PFA and MeOH fixation protocols should be compared.