COPII is more of a marker for exit sites on the ER than of a marker of the Golgi apparatus. The structure of the ER in cardiomyocytes is quite similar to what you see in your images. In my experience, methanol fixation is not optimal for membrane structures, for example lysosomes. However, methanol fixation preserves the cytoskeleton well. Therefore, in cardiomyocytes with a heavy cytoskeleton architecture, there might be more nonspecific staining than usual. In your case, however, the staining looks fine if COPII is regarded as an ER-associated marker. If you are still not satisfied, you may try various detergents instead of the commonly used Triton X-100. A good option for lysosomes is Brij56.

Rimma Sergeevna Kamentseva

Thanks for your answer. So do you mean that COPII in cardiomyocytes is not located around the nucleus but instead shows a striated pattern? In addition, when you use Brij56, what's the concentration and duration?

Chien-Wei Chang

My expertise lies more in the field of endocytosis than in that of the ER-Golgi system. It's known that the striated pattern is shown by smooth ER in skeletal myocytes. However, I don't know if the rough ER, where COPII-coated exit sites are located, exhibits the same pattern. It's worth reading more specific literature about the ER-Golgi membrane system in cardiomyocytes. Also, I think that the COPII pattern should be more punctate, but a lot of puncta may visually fuse to a striated-like pattern. So I don't rule out that possibility.

We fix cultured cells with 3.7% PFA in PBS for 15 minutes. Then, we permeabilize them with 0.05% Brij 56 in PBS for 15 minutes. The 10% Brij 56 stock solution is usually solid at 4°C, so it needs to be warmed to 56°C before dilution. After that, incubate with 1% BSA for 30 minutes, and then you can proceed with your antibodies.