Wheat germ agglutinin (WGA), a member of the lectin family that binds to N-acetyl-D-glucosamine and sialic acid residues found on the surface of cell membranes, has been used extensively to stain surface membranes, including t-tubules in cardiac myocytes (Savio-Galimberti et al. 2008). It has also been used as an indicator of the Golgi apparatus and nuclear core complexes (Iida & Page 1989; Miller et al. 1991). In this study, we have examined the effect of fixation on the staining of rabbit cardiac myocytes with WGA. Isolated rabbit atrial and ventricular myocytes were stained with an AlexaFluor 488-conjugated WGA. Images were acquired using a Leica SP5 confocal imaging system and excitation wavelengths of 488 nm and 590 nm with the aperture set to 1 Airy unit. Images were analysed using ImageJ: Binary images were calculated by thresholding images and the percentage of bright pixels within the cell including (‘%total area’) and excluding (‘%intracellular area’) the cell edge were calculated. Cells were fixed with 4% paraformaldehyde (PFA) for 10 min before incubation with WGA for 10 min, 30 min or 1 hr . The duration of incubation had no significant effect on the percentage total area or the percentage intracellular area stained. However, significant intracellular staining was observed in atrial and ventricular myocytes (atrial cells, 6.2±1.5%, n=13; ventricular cells, 9.0±1.5%, n=13; P>0.05, Mann-Whitney test). Visual inspection showed a staining pattern in ventricular cells consistent with t-tubules. On the other hand, the intracellular staining in atrial myocytes suggested binding of the WGA to intracellular organelles, such as the nucleus and Golgi. This suggested that the membranes had been permeabilized during fixation allowing WGA to enter. To investigate this possibility, cells were stained with WGA before fixation. With this technique, atrial myocytes showed significantly less staining inside the cell (0.1±0.04% of intracellular area, n=7), compared to ventricular myocytes (7.7±0.93%, n=9), p<0.001, Mann-Whitney test. Cells fixed before staining were compared with cells treated with 0.1% Triton-X to permeabilise the membrane, to assess whether permeabilization during fixation could explain the staining pattern observed. This method produced staining similar to non-permeabilised cells with 6.66±1.81% of the intracellular area stained in atrial cells (n=7), and 9.67±1.38% in ventricular (n=7), P>0.05 unpaired t-test. In summary, WGA is an important tool for staining the cell membranes, however the protocol used for staining must be considered. The current data suggests that fixing cells using PFA results in significant permeabilisation of the cell membrane and subsequent intracellular staining with WGA. Care should therefore be taken in the interpretation of images of cardiac myocytes stained using WGA.