1% protease XXIV–treated paraffin sections (2 µm thick) using rabbit anti-SLC12A1 Ab (catalog no.: AV41388; dilution, 1:100; Sigma-Aldrich, St. Louis, MO). Primary Abs were detected using appropriate secondary Abs, including polyclonal rabbit anti-rat immunoglobulins/biotinylated (catalog no.: E0468; Dako Denmark A/S, Glostrup, Denmark) and rabbit on rodent HRP-Polymer (catalog no.: RMR622; Biocare Medical, Concord, CA) for VCAM-1 and F4/80 and rabbit on rodent HRP-Polymer (catalog no.: RMR622; Biocare Medical) for AQP2 and NKCC2. Binding of Abs was visualized using β-amino-9-ethyl-carbazole
(Dako AEC + High Sensitivity Substrate Chromogen Ready-to-Use; catalog no.: K3461; Dako Denmark A/S) as substrate. Immunofluorescence (IF) for basement membrane protein laminin and AQP2 was learn more performed on formaldehyde/methanol/acetone–fixed cryosections of kidney tissue (3 µm thick) using rabbit anti-laminin (catalog no.: ab11575; dilution, 1:200; Abcam plc) and either rabbit anti-AQP2 (catalog no.: find more ab85876; dilution,
1:500; Abcam plc) or goat anti-AQP2 (catalog no.: ab105171; dilution, 1:500; Abcam plc). Double-labeling IF was performed on formaldehyde/methanol/acetone–fixed cryosections of kidney tissue (3 µm thick) using rabbit anti-laminin (catalog no.: ab11575; dilution, 1:200; Abcam plc) and goat anti-AQP2 Ab (catalog no.: ab105171; dilution, 1:500; Abcam plc) as 上海皓元 well as goat anti-AQP2 Ab (catalog no.: ab105171; dilution, 1:500; Abcam plc) combined with rabbit anti-AE1 (anion exchanger 1) Ab for staining of type A intercalated cells (dilution, 1:750[21]) or goat anti-AQP2 Ab (catalog no.: ab105171; dilution, 1:500; Abcam plc) combined with
an Ab against mouse pendrin for identification of non-type-A intercalated cells (guinea pig anti-pendrin; dilution, 1:500[21, 22]). Primary Abs were detected using the following fluorophore-conjugated secondary Abs: Alexa Fluor 594 Goat Anti-Rabbit IgG (immunoglobulin G; catalog no.: A-11037; Life Technologies, Carlsbad, CA); Alexa Fluor 488 Donkey Anti-Goat IgG (catalog no.: A-11055; Life Technologies); and Rhodamine Red-X-AffiniPure F(ab’)2 Goat Anti-Guinea Pig IgG (catalog no.: Jackson ImmunoResearch Europe Ltd., Newmarket, UK). Slides where counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1.5 µg/mL). Fluorescent-labeled ursodeoxycholic acid (UDCA; ursodeoxycholyl [UDC]/Nε-4-nitrobenzo-2-oxa-1,3diazol [NBD]/lysine; molecular weight, 684; kindly provided by Prof. Dr. Alan Hofmann, University of California, San Diego, La Jolla, CA) was injected into the portal vein of 3-day CBDL mice and sham-operated controls. After relaparotomy, the portal vein was cannulated using a 27-G needle. Thereafter, UDC/Nε-NBD/lysine (100 µmol per mouse solubilized in 200 mL physiologic saline solution) was given continuously over 10 minutes. Subsequently, kidneys were excised and frozen immediately in liquid nitrogen.