Oct embedding matrix

Mice were euthanized in a controlled CO₂ chamber (TEM Sega) and blood sampling was performed through cardiac puncture with a heparin-coated syringe with a 25 G needle. Blood was centrifuged at 300 g for 15 min and plasma was harvested and kept at −80 °C for later analysis. RBC were lysed in 5 mL of RBC lysing buffer for 5 min at rt and washed with 10 mL of PBS. This procedure was repeated 3 times and cells were resuspended in PBS. The left kidney was harvested and embedded in OCT embedding matrix (Cellpath) and snap-frozen in liquid nitrogen and kept at −80 °C for later analysis. For cell analysis, spleen and lymph nodes (cervical, brachial, and inguinal) were harvested in PBS and dissociated by mechanical disruption on a 40 μm cell strainer (Falcon, Corning). For splenocytes, RBC were lysed once in 5 mL RBC lysing buffer 5 min at rt and washed with 10 mL of PBS. Cell counts were assessed by trypan-blue staining on a Malassez hemacytometer and 1 to 5 million cells were used per FACS staining condition. For immunofluorescence analysis, spleen was harvested and embedded in OCT embedding matrix (Cellpath) and snap-frozen in liquid nitrogen, and kept at −80 °C for later analysis.

Animals were perfused through the left cardiac ventricle with a pre-fixative solution (1000 U/ml Heparin, 0.2% procain-HCl, 3.2% CaCl₂ and 0.18% NaCl) followed by the fixative (3% Paraformaldehyde (PFA)/PBS). After incubation in PFA for one hour, kidneys were placed overnight in 32% sucrose/PBS and subsequently embedded in OCT embedding Matrix (Cell Path, Newtown, Wales, United Kingdom) and frozen in liquid propane. Cryosections of 5 μm were mounted on slides (Superfrost Plus, Thermo Scientific) and blocked with 3% bovine serum albumin/PBS; the primary antibodies against MCT14 (1/1000), NKCC2 [25 (link)] (1/1000), Uromodulin [26 (link)] (1/400), NCC [27 (link)] (1/500) or NaPi-IIa [28 (link)] (1/400) were diluted in PBS and added to the cryosections for incubation over night at 4°C. Then, samples were washed twice with hypertonic PBS (18g NaCl/PBS), once with PBS and incubated with the secondary antibodies donkey anti-rabbit Alexa 594, donkey anti-sheep Alexa 488 and goat anti-guinea-pig Alexa 488 (1/1000, Invitrogen) during 2 hours at room temperature. After two consecutive washing steps with hypertonic PBS and once with PBS, cover slips were mounted with Glycergel (DakoCytomation, Baar, Switzerland). Fluorescence was detected with a Leica fluorescence microscope (Leica CTR600). Leica AF lite and Image J freeware programs were used to process the pictures.