All animal experiments were in accordance with the Animal Care Committee. An established model of murine renal IRI was used . In brief, CD-1 mice were anesthetized (60mg/kg sodium pentobarbital, ip). Kidneys and renal pedicles were exposed by bilateral flank incisions, and renal pedicles were dissected. A non-traumatic vascular clamp was applied across the left pedicle for 30min. The right kidney provided a sham control.
Paraformaldehyde, 16% (Thermo Scientific #28908)
Published methods were applied to examine kidneys from CD-1 mice . In brief, specimens were fixed for 30min in 4% paraformaldehyde, washed three times in PBS and paraffin-embedded, following standard procedures. Specimens were sectioned (4µm sections), placed on gelatin-coated slides and processed.
_Standard equipment for immunohistochemistry_
Glass slide jars (microwave-compatible)
Microwave, sample delimiting pen (DakoCytomation, #S2002) to draw a hydrophobic barrier
_Solutions for deparaffinization and hydration_
95% ethanol in phosphate buffered saline (PBS)
70% ethanol in PBS
50% ethanol in PBS
30% ethanol in PBS
_Antigen retrieval solution_
10mM Trisodium citrate pH 6.0 in water, supplemented with 0.5% (v/v) Tween-20
PBS containing 5% fetal bovine serum (FBS), 0.05% Tween-20 (v/v), 1mM NaN3
PBS containing 0.1% Triton X-100 (v/v), 2mg/ml bovine serum albumin, 1mM NaN3
Mouse anti-HuR (Santa Cruz; #sc-5261)
Rabbit anti-aquaporin-1 (Alpha Diognostics)
Cy3-conjugated anti-mouse IgG Jackson ImmunoResearch (715-165-150)
AlexaFluor®488-conjugated anti-rabbit IgG, Jackson ImmunoResearch (711-545-152)
Secondary antibodies were further purified by pre-adsorption to immobilized proteins prepared from mammalian culture cells.
4’,6-diamidino-2-phenylindole (DAPI; Sigma)