Fig. 9. AMF and CTX do not cointernalize. NIH-3T3 cells were coincubated with 50 µg/ml of AMF-FITC and 5 µg/ml of Alexa 594-CTX at 37°C for 5 minutes (A-F) or 30 minutes (G-J) prior to fixation with precooled methanol/acetone. The distributions of AMF-FITC, revealed with rabbit anti-FITC followed by Alexa 488 anti-rabbit antibodies (A and G), and of Alexa-594–CTX (B, H) were compared after five minutes with the smooth ER labeled with anti-AMF-R mAb and Alexa 647 anti-rat IgM antibodies (C) or at 30 minutes with the Golgi apparatus labeled with anti-GM130 mAb and Alexa 647 anti-mouse antibodies (I). Merged confocal images present AMF-FITC in green and either AMF-R (D) or Alexa 594-CTX (E) in red. Triple merges show AMF-FITC in green, Alexa 594-CTX in red and either AMF-R (F) or GM130 (J) in blue. Bar, 8 µm.