Fig 1: Nearest distance of two kinds of membrane proteins and the effect of fixatives.a EpCAM image of a living cell. b E-cadherin image of the same cell as a. c Superimposed image of EpCAM (a green) and E-cadherin (b red). d EpCAM image after 4% PFA treatment. e E-cadherin image of the same cell as d. f Superimposed image of EpCAM (d, green) and E-cadherin (e red). g EpCAM image after 2% GA treatment. h E-cadherin image of the same cell as g. i Superimposed image of EpCAM (g green) and E-cadherin (h red). j EpCAM image after 100% EtOH treatment. k E-cadherin image of the same cell as j. l Superimposed image of EpCAM (j, green) and E-cadherin (k red). m Distribution of the average nearest distance per cell. n = 14 (living), 15 (PFA), 13 (GA) and 14 (MeOH). Red lines indicate mean values. Asterisks (*, ** and ***) denote statistical significance (p < 0.05, 0.01 and 0.001, two-sided t test). n Time series during fixation. EpCAM (green) and E-cadherin (red) are presented. Arrowheads indicate the same molecule. The scale bar is 1 µm. 2% GA was added at 0 s. o Time-lapse change of the distance from the E-cadherin indicated in n to the nearest EpCAM. The dotted line indicates the time point of GA addition. p Model of the behavior of membrane proteins during fixation. Before adding fixatives, membrane proteins approach and move apart from each other. However, after adding a fixative, proteins cannot move apart once they make contact.