Roti histokitt 2

Formalin fixed paraffin embedded kidney tissue of healthy mice and mice after IRI was sectioned at 4 µm and stained with Periodic Acid-Schiff (Sigma-Aldrich) using a standard protocol. Finally, slides were dehydrated, cleared and mounted in anhydrous mounting medium Roti-Histokitt II (Carl Roth). Stainings were evaluated for the number of tubular casts in six high power fields (HPFs) by one blinded examiner.

Methacarn-fixed, paraffin-embedded tissue sections were used for immunohistochemistry (using the avidin–biotin complex (ABC) method). For dewaxing, the sections were placed for 10 min in xylene, followed by a descending alcohol series and immersion with distilled water for 5 min. For KLOTHO and fibrinogen staining, specimens were heated in a 10 mM sodium citrate buffer (pH 6.0) in a microwave for 5 min. Endogenous peroxidase was inactivated by incubation with 3% hydrogen peroxide. After washing, the sections were incubated with a blocking solution (10% FCS, 1% BSA in 1× Tween–PBS) for 30 min at room temperature, and a primary antibody (goat anti-mouse KLOTHO, R&D Systems, AF1819; goat anti-mouse Fibrinogen, Santa Cruz, 18032) was added overnight at 4 °C diluted in 1% FCS plus 1× Tween–PBS. After incubation with a peroxidase-labeled secondary antibody (Jackson Immuno Research, 705-035-147, 1:500) for 1 h at room temperature, the sections were developed using a diaminobenzidine substrate and counterstained with a hematoxylin solution. Subsequent dehydration was achieved using ascending alcohol series (70% ethanol, 80% ethanol, 96% ethanol, and 100% ethanol). Finally, washing was performed twice for 5 min in Histo-Clear (Carl Roth, Karlsruhe, Germany). Embedding was performed with a ROTI-Histokitt II (Carl Roth, Karlsruhe, Germany), according to the manufacturer’s instructions.

FFPE tissue was cut into 2 μm sections on a microtome and de-paraffinized. After antigen retrieval slides were incubated with primary antibody (the primary antibodies used are listed in Table S3) over night at 4 °C and washed with Tris-HCL (Tris hydrochloride) buffer (pH 7.5) followed by a secondary antibody. Antibodies were detected with the Vectastain Elite ABC (avidin-biotin complex) kit (Vector) using DAB (3,3′-diaminobenzidine) (Vector Laboratories and Dako) for brown stainings or AEC (3-Amino-9-ethylcarbazole) (Thermo Fisher Scientific) for magenta stainings. The slides were counterstained with hematoxylin (Vector Laboratories) and mounted with Roti®-Histokitt II (Carl Roth, Germany). Images were captured on an Axioplan2 imaging microscope (Carl Zeiss) equipped with an AxioCamHRc Camera (Carl Zeiss) or slides were scanned with a Vectra Polaris imaging system (PerkinElmer, Hopkinton, MA, USA) and quantified by ImageJ software.