Hello Jonas Rybnicek

In a 3D structure, the fixative must diffuse through the outer cell layers and the extracellular matrix to reach the cells in the core. Therefore, a longer incubation time (like 10–15 minutes) is necessary to ensure complete and uniform fixation. Moreover, cold temperature is vital for 3D fixation. It slows down the denaturation process, which helps prevent over-fixation of the outer cell layers before the inner cells are properly fixed. This maintains the structural integrity of the delicate 3D architecture, preventing shrinkage.

On the other hand, in a monolayer, methanol has a very short distance to travel to fix every cell. A shorter incubation time (e.g., 5 minutes) is sufficient to denature and precipitate proteins throughout the cell.

So, for 3D culture, the standard method uses a longer fixation time (10-15 mins) and a colder temperature (-20°C) to ensure the fixative fully penetrates the entire structure, which is not necessary for a flat, 2D monolayer.

Best,