Alexa 488 and 546 dye labeled antibodies

Mouse kidneys were fixed by perfusion through the left ventricle with 0.2 M periodate lysine and 2% paraformaldehyde in PBS. Tissue samples were soaked for several hours in 20% sucrose in PBS, embedded in Tissue-Tek OCT Compound (Sakura Finetechnical), and snap-frozen in liquid nitrogen. Goat anti-AQP2 (C-17, Santa Cruz, sc-9882; 1:500) was used as the primary antibody. The mpkCCD cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X/PBS. The filters were detached from the holders and incubated with the following primary antibodies: goat anti-AQP2 (N-20; 1:500), rabbit anti-AQP2 (phospho S269; 1:500), and Acti-stain 555 phalloidin (Cytoskeleton, # PHDH1-A; 7:1,000). Alexa 488 and 546 dye-labeled antibodies (Molecular Probes; 1:200) were used as the secondary antibodies. The samples were mounted with Vectashield/DAPI (Vector Laboratories). Immunofluorescence images were obtained using the LSM510 Meta (Carl Zeiss). The fluorescence intensities of AQP2 in mouse kidneys were quantified using Zen 2009 software. The fluorescence intensities of AQP2 in the mpkCCD cells were quantified using ImageJ software.

HUVECs were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X/PBS. Rabbit anti-phospho-PKA substrate (dilution, 1:200; #9624; Cell Signaling Technology), mouse anti-Myc-Tag (dilution, 1:200; #9B11; Cell Signaling Technology), rabbit anti-HA-tag (dilution, 1:200; #C29F4; Cell Signaling Technology), mouse anti-VE-cadherin (dilution, 1:200; #sc-9989; Santa Cruz), and rabbit anti-Halo-tag (dilution, 1:200; #G9281; Promega Corporation) were used as primary antibodies. Alexa 488 and 546 dye-labeled antibodies (dilution, 1:200; Molecular Probes) were used as the secondary antibodies. The actin cytoskeleton was detected using Acti-stain™ 555 phalloidin (dilution, 1:1000; #PHDH1-A; Cytoskeleton Inc.). The samples were mounted with ProLong™ Glass Antifade Mountant with NucBlue™ (P36981; Invitrogen Corporation). Immunofluorescence images were obtained using a Leica SP-8 confocal microscope. The intensity of stress fibers was quantified using ImageJ software (https://imagej.nih.gov/ij/).