Mayer s hematoxylin solution

The artery specimen were embedded with Tissue Tek (Tissue-Tek O.C.T Compound, Sakura) and liquid nitrogen on cryomolds. The membrane slides (MembraneSlide 1.0 PEN, Carl Zeiss Microscopy GmbH) were pretreated with UV light for 15 min. Frozen sections of 8 μm were cut and mounted on the slides. The slides were stored at − 80 °C. To enhance the visibility of the EC, the frozen sections were stained with hematoxylin (Mayer’s hematoxylin Solution, Sigma Aldrich). To prevent the slides from defrosting, the hematoxylin, ethanol solutions and xylene were precooled. The slides were stained and dehydrated with graded ethanol solutions (75, 95 and 100%) ending with xylene. Afterwards the slides were allowed to dry for a maximum of 5 min in the hood. The stained slides were stored at − 80 °C.

Independent evaluation of all renal biopsies was performed by two renal pathologists, who were blinded to data assessment and data analysis. Within each renal biopsy specimen, each glomerulus was separately evaluated for present crescents, global sclerosis, and necrosis. The percentage of glomeruli with any of these pathologies was calculated as a fraction of the total number of glomeruli in each renal biopsy. Renal biopsies were scored in accordance with the current version of the Banff score for allograft pathology, as recently described [47 (link),48 (link)]. Kidney sections (formalin-fixed, paraffin-embedded) were deparaffinized in xylene and then rehydrated in ethanol containing distilled water. Utilizing antibodies against C3c (1:10,000, A0062, Agilent Dako, Santa Clara, CA, USA) and C4d (1:50, 503-17344, Zytomed, Berlin, Germany), tissue sections were stained. According to the manufacturer’s protocol, labeling was conducted using the Novolink^TM Polymer Detection System (Leica Biosystems, Wetzlar, Germany). Nuclear counterstaining was performed using Mayer’s Hematoxylin Solution (Sigma, St. Louis, MO, USA). C3c and C4d deposits in the glomerular tuft, interlobular arteries, peritubular capillaries, and venules were evaluated for presence, as recently described [12 (link),41 (link)].

Five-micrometer-thick paraffin-embedded sections were deparaffined with toluol followed by rehydratation. The slides of each group were incubated for 5 min in unmasking buffer (citrate acetate 1.8 mm, sodium citrate 8.2 mm, pH 6.0) at 86 C. Then the slides were incubated with 0.3 U/μl terminal deoxynucleotidyl transferase (Euromedex, Mundolsheim, France), 6.7 mm biotin-11-dUTP (Euromedex), and 26.7 mm dATP (Promega, Charbonnières, France) in terminal deoxynucleotidyl transferase buffer 1 h at 37 C. For the negative control, the enzyme was omitted. Extravidin alkaline phosphatase conjugate (dilution 1:100; Sigma-Aldrich) was added onto the slides for 25 min. Sigmafast FastRed TR/Naphthol AS-MX (Sigma-Aldrich) was used as the substrate according to the manufacturer’s instructions. Counterstain was performed with Mayer’s hematoxylin solution (Sigma-Aldrich) for 30 sec. In each testis, at least 100 random seminiferous tubules were counted. Results are expressed as the number of TUNEL-positive cells per 1000 seminiferous tubules. We perform the cell count (for TUNEL) in two to three independent experiments with at least 5 mice per group. In addition, for each male, counting was made on 2 non following slides.