Ab168387

The cells were fixed with ice-cold methanol for 10 min and then permeabilized with PBS (-) containing 0.1% Triton X-100 at room temperature (RT) for 15 min. After blocking nonspecific reactivity with 1% BSA in PBS(-) at RT for 1 h, the cells were incubated at 4 °C overnight with antibodies against Na⁺/K⁺-ATPase (No. 05-369, Merck), ZO-1 (No. 33-9100 and No. 61-7300, Thermo Fisher Scientific), AQP1 (ab168387, Abcam), SLC4A11 (HPA018120, Merck), NBCe1 (sc-515543, Santa Cruz Biotechnology Inc.), NHE1 (ab67314, Abcam), MCT1 (NBP1-59656, Novus Biologicals), and MCT4 (ab234728, Abcam). After washing with PBS (-), the cells were incubated for 1 h with fluorescent Alexa-Fluor secondary antibodies. The cell nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI) (No. 340-07971, Dojindo, Laboratories). The signal was detected using a fluorescence microscope (BZ-9000; Keyence Corp., Osaka, Japan).

Each TMA contained 17 patients’ tissues including 2 cores of tumor tissue and 1 core of paracancerous tissue for one patient. The core diameter of TMA was 1.6 mm. The immunohistochemical staining was performed on a Leica BOND-MAX autoimmunostainer (Leica Instrument Co., Ltd.). The tissue chips were first cut into 4-μm-thick slices using a microtome, attached to slides, dried, and baked at 60°C for 24 h before use. The prepared slices were dewaxed with xylene, rehydrated with different concentrations of ethanol (100% and 95%), rinsed with distilled water, and repaired with heat-induced antigen. Finally, the antigen was detected by an automatic staining machine (Benchmark) and Bond Polymer Purification Detection Kit (LeicaInstrumentCo., Ltd.). The primary antibodies (Abcam) were diluted as follows: anti-AQP1 (ab168387, 1:1,000), anti-AQP3 (ab125219, 1:500), and anti-AQP5 (ab92320, 1:200).

Protein was obtained after extracting RNA from the samples with Trizol reagent (Invitrogen). Protein was extracted from the same sample, precipitated and washed accordingly to the manufactures protocol. The protein was solubilized in lysis buffer with 1% sodium dodecyl sulfate. β-mercaptoethanol was added as a reducing agent making up 5% of the solution. Protein concentration was determined in triplicates on NanoDrop Spectrophotometer (NanoDrop 2000c, Thermo Scientific, Waltham, MA). The proteins were separated using 4–10% bis–tris gel (Invitrogen) under gel electrophoresis and transferred to a polyvinylidene difluoride membrane using an iblot machine (Invitrogen). The membrane was blocked and incubated in primary antibody overnight diluted 1:1000 for Aqp1 (ab168387, Abcam, Cambridge, UK), Lamin-B1 (ab65986, Abcam) and Nkcc1 (14581, Cell Signaling Technology, Danvers, MA). Membranes were incubated in secondary antibody diluted 1:10,000 and the protein bands were detected using chemiluminescent (Amersham ECL, GE Healthcare, Chicago, IL). The reaction was captured on luminescent image analyzer (LAS-4000 Luminescent Image Analyzer, Fujifilm, Tokyo, Japan). Protein band was quantification using ImageJ (NIH, USA). The relative protein expression is related to the loading control.

For immunofluorescence staining of kidney tissue, the samples were dewaxed and dehydrated, and then they were permeabilized and blocked. Subsequently, samples were incubated with primary antibodies against CD4 (Abcam, ab183685), CD8 (Abcam, ab217344), Aqp1 (Abcam, ab168387), Lrp2 (Abcam, ab76969), Ki67 (Abcam, ab15580), CD86 (Abcam, ab220188), α-SMA (Proteintech, 14395-1-AP), CD86 (Abcam, ab220188), and MHC Class II (Abcam, ab23990) at 4°C overnight and fluorescence secondary antibodies. Samples were stained with Hoechst. For IHC staining, experimental procedures were mentioned at the part of immunofluorescence staining before incubation with HRP-labeled antibody. The samples were treated with DAB assay kit. All images were scanned by using confocal microscope.

Frozen tissue sections stored in −80 °C were thawed in RT to be fixated with 3% freshly made paraformaldehyde in TBS for 10 min in RT. Tissues were then permeabilized for 10 min in TBS+ 0.1%Triton-X100, rinsed three times in TBS for 5 min/ rinse. Blocking with 2% bovine serum albumin in TBS for 2 h was performed before the tissues were incubated with primary antibodies overnight at 4 °C. After rinsing with 3 × 5 min with TBS the tissues were incubated with the secondary antibodies donkey anti-mouse immunoglobulin G (IgG)-AlexaFluor 568 (1:500 Molecular Probes) and donkey anti-rabbit IgG-AlexaFluor 647 (1:500 Molecular Probes) for 1 h at RT in darkness. DNA was counterstained with DAPI (Molecular Probes) and slides were mounted with Prolong Gold (Molecular probes). Tissues were stained with antibodies against SPINK1 (1:50, H00006690-M01, 4D4, Novus), TFF3(1:200, HPA 035464, Sigma), SPON2 (1:100, A-10, st cruz), PGC (1:50, NBP1-91011, Novus), NPY (1:100, ab48789, Abcam), Aquaporin (1:100, ab168387, Abcam), NR4A1 (1:100, ab48789, Abcam), ACPP (1:100, Biologicals), P63(1:150, ab53039, Abcam), Vimentin (1:150, ab8069, Abcam). Fluorescence images were obtained with a Zeiss LSM 780 inverted confocal microscope, using a Plan Apochromat 20 × /NA (numerical aperture) 0.7 objective. Tiled images were acquired from optical sections of 5 micrometer.