Use of lectin histochemistry to diagnose Sida carpinifolia (Malvaceae) poisoning in sheep

by standard histological methods and stained with haematoxylin and eosin. Microscopically, there was distension and vacuolation of Purkinje cells in the cerebellum (Fig 1) and of neurons in the cerebral cortex, thalamus, mesencephalon and spinal cord. Cytoplasmic vacuolation was also present in the epithelium of the pancreatic acinus and renal tubules, follicular epithelium of the thyroid, in hepatocytes, and in macrophages of the lymphoid tissues. Axonal spheroids were observed in the brain and spinal cord, particularly in the granular layer of the cerebellum. Lectin histochemistry was conducted on formalin-fixed, paraffin-embedded sections of the cerebellum and pancreas. The lectins used were obtained commercially (E-Y Labs) and are listed in Table 1. After deparaffinisation, the sections were incubated in 0·3 per cent hydrogen peroxide (H2O2) in methanol for 30 minutes at room temperature, rinsed several times in 0·01M phosphate-buffered saline (PBS), pH 7·2, and treated with 0·1 per cent bovine serum albumin in PBS for 15 minutes. Subsequently, they were incubated with biotinylated lectins for one hour, followed by incubation with avidinbiotin-peroxidase complex (Vector Labs) for 45 minutes. The peroxidase was marked by incubation for four to 10 minutes with buffered Tris-HCl, 0·05M, pH 7·6 solution containing 0·02 per cent diaminobenzidine and 0·05 per cent H2O2.All the sections were counterstained with Mayer’s haematoxylin. Each lectin was used at a dilution of 30 μg/ml in PBS, except for Arachis hypogaea (Table 1), which was applied at 10 μg/ml. As controls for the lectin histochemical procedure, the lectins were omitted or blocked by incubating them with their blocking sugars (0·1 to 0·2 per cent in PBS) for one hour at room temperature before application to the sections (Leathem 1986). The results of the lectin-binding patterns of affected and control sheep are summarised in Table 2. The cytoplasm of