Immunofluorescence

2.6.1 Human renal tissue collection and preparation

Human renal tissue was collected, prepared, and supplied to this investigation by Dr Shabbir Moochala (Institute for Cell and Molecular Biosciences, Newcastle University). Tissue was collected from the normal pole of nephrectomised specimens at the Freeman Hospital, Newcastle upon Tyne. Ethical approval was obtained from the Sunderland Local Research Ethics Committee. Kidney tissue was obtained after informed consent from patients undergoing nephrectomy for renal cell carcinoma subject to the following exclusion criteria: serum creatinine > 140 μmol/l, or any evidence of renal scarring.

Tissue was immediately removed to a renal preservation solution (Ahmad et al., 2006) at 4 °C and transported to the laboratory on ice. The solution consisted of: 140 mM sucrose, 42.3 mM Na2HPO4, and 26.7 mM NaH2PO4 in distilled water. This was pH corrected to 7.4, then autoclaved, aliquoted into sterile containers, and stored at 4 °C until use.

Human tissue on ice was cut into blocks of 5 mm x 5 mm in a Class II ventilation hood before being embedded, as a small volume of tissue enabled quicker and more even

freezing. Tissue blocks were embedded in OCT embedding medium (RA Lamb Ltd., Eastbourne, UK/Thermo Fisher Scientific) and then snap-frozen, attached to a cork mounting block, by immersion in liquid nitrogen-chilled isopentane (2-methylbutane). Once frozen, tissue blocks were stored in a sealed container at -80 °C.

2.6.2 Mouse renal tissue collection and preparation

Normal mouse renal tissue was supplied by Newcastle University Comparative Biology Centre and was immediately immersed in DMEM/HAM’s F12 1:1 vol/vol at 4 °C to preserve the tissue for transportation to the laboratory on ice. As with the human tissue preparation, mouse tissue was cut into blocks of 5 mm x 5 mm before being embedded. Tissue blocks were embedded in OCT embedding medium (RA Lamb/Thermo Fisher Scientific) and then snap-frozen, attached to a cork mounting block, by immersion in liquid nitrogen-chilled isopentane (2-methylbutane). Once frozen, tissue blocks were stored in a sealed container at -80°C.

2.6.3 Tissue sectioning

Blocks of embedded tissue were removed from storage and allowed to acclimatise in a Cryostat chilled to -20°C before tissue sections of 6 µm thickness were cut. Cut sections were immediately applied to Superfrost Plus microscope slides (VWR International Ltd., Leighton Buzzard, UK) or to standard Superfrost microscope slides (Thermo Fisher Scientific) that had been pre-coated with 3-aminopropyltriethoxysilane (APTES) (Sigma- Aldrich). Tissue slides were stored at 4°C until they were prepared for immunofluorescence studies.

2.6.4 Preparation of tissue and cells for immunofluorescence studies

Tissue sections attached to slides were ringed with a hydrophobic pen before being fixed in cytoskeletal fixative solution for 10 min. Cytoskeletal fixative consisted of: 4% paraformaldehyde (Sigma-Aldrich), 100 mM PIPES pH 6.8 (Sigma-Aldrich), 2 mM EGTA (Sigma-Aldrich) and 2 mM MgCl2 in H2O. Sections were then washed with 150 mM Tris HCl to quench fixative before permeabilisation of tissue using 0.2% Triton X100 for 10 min. Sections were washed with Phosphate-buffered saline with Tween20 (PBS/T) three times and blocked with 3% horse serum for 1 hour at room temperature. PBS/T consisted of: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 0.05% Tween20 in water. Sections were then washed another three times with PBS/T before treatment with primary antibody (Table 2.7) at the appropriate dilution in block solution for 2 hours at room temperature or overnight at 4°C. After primary antibody incubation, sections were washed and then blocked again, as before, by incubation with 3% secondary antibody host serum for 1 hour, with mild agitation, and at room temperature. Secondary antibody incubation was under the same conditions as for primary. Finally, sections were washed three times in PBS/T and once in ultrapure H2O before coverslips were affixed over the sections using fluorescence mounting medium (Dako UK Ltd., Ely, UK). Slides were kept in the dark and at room temperature until mounting medium was set, after which, slides were stored in the dark at 4°C.

Antibody Name Type Epitope Dilution Anti-DYKDDDK

(Chemicon)

Monoclonal IgG DYKDDDK 1:1000

Anti-AQP2 (C-17)

(Santa Cruz) Polyclonal AQP2 C-terminus 1:250

Anti-SGK1D 3253

(Cambridge Biosciences) Polyclonal CNHANILTKPDPRTFWTNDDP 1:100 Anti-γ tubulin (Abcam) Monoclonal IgG1 γ tubulin 38-53 1:200 Anti-acetylated α tubulin

(Sigma-Aldrich)

Monoclonal IgG2b Acetylated α tubulin Lys 40

1:200 Anti-ZO-1

(Zymed (Invitrogen))

Monoclonal IgG1 k Human ZO-1 334-364. 1:200 Anti-Calnexin (H-70)

(Santa Cruz) Polyclonal Human Calnexin 1-70. 1:100 Table 2.7 Primary antibodies used in immunofluoresence studies.

Cells grown on coverslips were treated in exactly the same way as tissue, except that, after staining, they were attached face-down onto Superfrost microscopy slides (Thermo Scientific). In some experiments, the actin cytoskeleton of cells was stained using Alexa Fluor 633 Phalloidin (Molecular Probes, Invitrogen) at a concentration of 1:500. Secondary antibodies for all experiments were from Molecular Probes, Invitrogen and were used at a concentration of 1:300 for 2 hours. For tissue staining, secondary antibodies raised in donkey were used, whereas for cells, secondary antibodies raised in goat or rabbit were used. Nuclei were stained by using Hoechst 33258 at a concentration of 1:1000 for 10 min. The following fluorophores and their representative colours were used: Hoechst (blue), Alexa Fluor 488 (green), Oregon Green 488 (green) Alexa Fluor 568 (red), Alexa Fluor 633 (red).

2.6.5 Microscopy

Slides were imaged using either a Leica TCS SP2 UV Upright Confocal System or a Zeiss LSM 510 META laser scanning confocal microscope. The lenses used for each system were a 63x HCX PL APO oil immersion lens and a Plan-Neofluar 40x/1.3 Oil Ph3 lens, respectively. Images where nuclei are shown were taken with the Leica microscope and consist of collapsed Z-stacks. Images that are two-channel only were taken using the Zeiss microscope and are single Z-sections. Images were collected sequentially to avoid emission signal bleed-through. For multiple slides from a single experiment channel settings were kept the same except for slight alterations of the gain and offset to reduce the appearance of non-specific staining. Z-section images were captured at ~1 µm intervals. For each experiment, positive signals were verified by comparison with controls.