Confocal 710

We used a Zeiss confocal 710, 880 or 900 for confocal microscopy. The same microscope was used for each imaging experiment, and identical imaging settings were used for all settings acquired by the blinded investigator. For quantification of amyloid in lymph node, we imaged regions of lymph node in draining regions based on CD31/LYVE1 staining and imaged at 425.10 µm² (1.204 pixels per µm), at 11 µm z-stacks at 2-µm step sizes. For quantification of amyloid in the prefrontal cortex, the region to be imaged was selected based on Hoechst reference and comparison with the mouse brain atlas, then we imaged a region of 319.45 µm² (3.2055 pixels per µm) using a 30 µm z-stack imaged at 1-µm step sizes. To ensure consistency and unbiased imaging by the blinded investigator, we used the Hoechst channel to set the upper and lower boundaries of each z-stack. Zeiss ZEN Blue (v3.3.89) (Carl Zeiss Microscopy) was used for image acquisition. For data analysis, Fiji image processing software (v1.54) (NIH) and Imaris (v9.1) (Oxford Instruments) were used.

For confocal microscopy experiments, HEK293 or MDCK cells were grown on glass coverslips and transiently transfected with Iqgap1, Slc26a4 (pendrin), or both expression vectors. The coverslips were fixed 48 h later with 4% paraformaldehyde 48 h after transfection. Fixed cells were labeled with IQGAP1 and SLC26A4 antibodies. The slides were observed using a Zeiss confocal 710. Z-stack images were obtained with LSM 5 Image software.

Cover slips carrying neuronal cultures were fixed with 4% PFA and mounted onto an object slide with PVA-DABCO mounting media (Sigma). Images were acquired on a Zeiss Confocal 710 with oil immersion 63x objective with 1.4 N.A. Images stacks were captured at 1024 x 1024 with an axial spacing of 6 micrometers Presented images are maximum intensity projections of three consecutive z-planes. All images are shown in 8 bit.