Envison system

After deparaffinization of the sections, antigen retrieval was performed by microwaving in 10 mmol/L citrate buffer (pH 6.0) for p-Akt, p-S6 and LC3 staining, and by heating in Tris-ethylenediaminetetraacetic acid buffer (pH 9.0) with a pressure cooker for p-mTOR and Ki-67 staining. After blocking the endogenous peroxidase, the sections were incubated overnight at 4°C with anti-p-mTOR (Ser2448) (1∶20; Santa Cruz Biotechnology, Inc.), anti-p-Akt (Ser473) (1∶25), anti-p-S6 (1∶200), anti-LC3 (1∶100; Nanotools) and anti-Ki-67 (pre-diluted) antibodies. Then the sections were incubated with the secondary antibody conjugated to the peroxidase-labeled polymer, EnVison+system (DakoCytomation). Color development was performed using DAB, and the sections were counterstained with hematoxylin. Control sections were evaluated by substitution of the primary antibodies with nonimmunized serum without signal detection.

Tissues (liver and kidney) were collected and fixed in 4% paraformaldehyde and then embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E). Tissue injury was examined by light microscopy. Immunohistochemical staining, using L. interrogans strain 56606v-specific rabbit antiserum prepared in our lab, was performed using the EnVison system (Dako, Glostrup, Denmark).