Aquaporin 2

Cryosections (7 μm) were cut using a cryostat from the frozen blocks of Ren1d-Cre; mT/mG mouse kidneys and subsequently subjected to the immunostaining for aquaporin 2 (1:200; Santa Cruz Biotechnology) and renin. Kidney sections from Ren1d-Cre;mT/mG mice were screened for the presence of GFP+ in collecting duct epithelial cells based on co-localization with AQP2 immunostaining in both diabetic and non-diabetic anmals using Fiji software (NIH). A Pearson’s correlation coefficient was used to quantify the colocalization parameters. Moreover, a large image analysis of the GFP and AQP2 colocalized area per total area was performed to assess any differences between kidneys of control and diabetic Ren1d-Cre;mT/mG mice.
In addition, kidney sections were assessed for the presence of GFP+ cells in control and diabetic Ren1d-Cre;mT/mG mice within the parietal glomeruli and glomerular tuft and quantified as as percentage of glomerular GFP positivity.

Immunofluorescence staining of tissue section was conducted as described previously^{2 (link)}. Confocal images were acquired in multichannel mode using a Zeiss Pascal 5 confocal laser microscope using the following laser and filter combinations: 488nm argon laser and BP 505–530nm filter; 543nm helium-neon laser and BP 560–615nm filter; and 633nm helium-neon laser and LP 650nm filter.
Control immunostaining with pre-immune serum or normal IgG of the same species as the primary antibody were performed for each experiment.
Human kidney biopsy specimens were embedded in OCT, 4 μm frozen sections were cut in a Cryostar 50 cryostat, and staining was performed using antibodies against PCSK9 (Cayman Chemical) and Aquaporin-2 (Santa Cruz).