Fig 4.

(a to i) Double fluorescence staining for VM (green) and GM (red) in the proximal tubule cells of the kidney of rats administered with both drugs. Rats were i.v. injected with a mixture of VM (33.3 mg/kg) and GM (16 mg/kg) and then sacrificed 12 h later. This was carried out in essentially the same manner as that used in the ICC except that a mixture of Alexa Fluor 488-labeled goat anti-mouse IgG and Alexa Fluor 555-labeled goat anti-rabbit IgG was used as the second antibody. The first antibody of the culture supernatant of the AVM-113 MAb was used at a dilution of 1:60, and predigestion of specimen sections was performed at 0.004% protease at 30°C for 15 min (a to c), 1 h (d to f), or 2 h (g to i). (a to c) Strong green fluorescence for VM is in both the microvilli and the cytoplasmic granules of the proximal tubule cells, and red fluorescence for GM is weak at the bottom of the microvilli and strong in the cytoplasmic granules. The merged image shows green fluorescence in the microvilli and yellow fluorescence in the cytoplasmic granules. (d to f) Strong green fluorescence is in both the nuclei and the cytoplasmic granules, and red fluorescence is in the cytoplasmic granules. The merged image shows green fluorescence in the nuclei and yellow fluorescence in the cytoplasmic granules. Note that no microvilli were seen in the cells as a result of the prolonged protease digestion. (g to i) Green fluorescence is in most of the cytoplasmic granules, and red fluorescence is in most of the cytoplasmic granules as well as in the three swollen cells. The merged image shows yellow fluorescence in most of the cytoplasmic granules and red fluorescence in the three swollen cells, possibly a part of the distal tubule cells.