Bx51 clinical microscope

At 6 days of transplantation, a morphological and immunohistochemical study was performed in five Lewis rat recipients to identify the type of graft rejection. Formalin-fixed tissue was embedded in paraffin. Sections (3-μm thick) mounted on xylene glass slides (Dako, Carpinteria, CA) were used for immunohistochemistry. After antigen retrieval had been carried out, endogenous peroxidase blocking for 10 min in 3% hydrogen peroxide (Merck, Darmstadt, Germany) was performed before primary antibody incubation. The primary antibody, rat anti-C4d (Hycult Biotech, PA), was incubated overnight at 4°C. Envision system-specific anti-rabbit secondary antibody labeled with horseradish peroxidase polymer (Dako, Glostrup, Denmark) was applied for 1 h. All sections were counterstained with Mayer hematoxylin. Immunohistochemical procedure was performed at the same time to avoid possible day-to-day variations in staining performance. All images were acquired using an Olympus BX51 clinical microscope and DP70 digital camera and software (Olympus, Tokyo, Japan).
A renal pathologist evaluated hematoxylin/eosin, periodic acid Schiff and C4d stains to evaluate renal damage.

Paraffin-embedded renal sections (3-μm-thick) were stained with periodic acid-Schiff (PAS). The quantification of tubular vacuolization, casts and cysts were assessed in 20 non-overlapping high-power fields (X40). All images were acquired using an Olympus BX51 clinical microscope and DP70 digital camera and software (Olympus, Tokyo, Japan).