Biotinylated mouse anti rabbit secondary antibody

Indirect immunoperoxidase staining was performed on formalin-fixed tissue. Briefly, 4-μm-thin tissue sections were deparaffinized in Xylene (VWR, Darmstadt, Germany) and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase (Fischar, Saarbruecken, Germany). Sections were incubated overnight at 4°C with primary antibodies diluted in 1% BSA/TBS. The sections were washed repeatedly in TBS before incubation with biotinylated mouse anti-rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) diluted in 1% BSA/TBS for 1 h at room temperature. The ABC kit (Vector, Burlingame, CA, USA) was used for signal amplification, and 3,3′-diaminobenzamidine (Sigma-Aldrich, St Louis, MO, USA) was used as a chromogen. Slides were counterstained with hematoxylin (Sigma-Aldrich), dehydrated, and covered with Histomount (National Diagnostics, Atlanta, GA, USA). Periodic acid Schiff (PAS) staining was performed for the assessment of glomerulosclerosis. Images were acquired with an Axiovert 200 M microscope/EC Plan-Neofluar ×40/1.3 oil immersion or C-Apo ×63/1.20 water immersion objective equipped with a charge-coupled-device camera (all from Carl Zeiss MicroImaging GmbH, Jena, Germany). Images were further processed using ImageJ/Fiji software version 1.46 (NIH, Bethesda, MD, USA).

Paraffin-embedded sections were deparaffinized in Xylene (VWR, Darmstadt, Germany) and rehydrated in decreasing concentrations of ethanol. Heat-induced antigen retrieval was performed in 10mM Tris 1mM EDTA 0,005% Tween buffer, pH9.0 for 15. Peroxidase blocking was performed in 3% hydrogen peroxidase (Roth, Karlsruhe, Germany). After incubation in primary antibody (anti-Aldh1a1 antibody, ab52492; anti-Asah1 antibody, ab74469 Abcam, Cambridge, UK) 1:200 in TBS 1% BSA at 4°C overnight, sections were washed in TBS and incubated in biotinylated mouse anti-rabbit secondary antibody (Jackson Immuno-research, West Grove, USA) 1h at room temperature. For signal amplification the ABC Kit (Vector, Burlingame, CA, USA) was used before applying 3,30-diaminobenzamidine (Sigma-Aldrich, St Louis, USA) as a chromogen. Hematoxylin was used for counter-staining. After dehydration slides were covered in Histomount (National Diagnostics, Atlanta, USA).
For the assessment of age-related histologic alterations periodic acid Schiff staining was used.