Detection of PrP^sc on Lymphoid Tissues from Naturally Affected Scrapie Animals: Comparison of Three Visualization Systems

Abstract

S U M M A R YWe assessed three different visualization systems used routinely in research and diagnosis of transmissable spongiform encephalopathies (TSEs) to demonstrate whether the methodology applied to immunohistochemical (IHC) examination may alter the results concerning detection of prion protein (PrP sc ) in the lymphoreticular system (LRS): avidin-biotin-peroxidase (Vectastain ABC kit; Vector), Envision (DAKO), and catalyzed signal amplification (CSA; DAKO). The study aimed to determine which of these showed the highest sensitivity, with the hope of providing an accurate tool for pathogenesis and preclinical diagnosis research in TSEs. Histological sections from palatine tonsils, spleen, GALT (ileum and ileocecal valve), and lymph nodes from sheep belonging to a Spanish scrapie-positive flock were processed by IHC using L42 as primary antibody. As substrate chromogen, diaminobenzidine was used, and all slides were subjectively assessed by light microscopy. A further study using an image analyzer software system was carried out to confirm that the conclusion provided by microscopic examination was objective. The CSA system showed the highest sensitivity in all cases, increasing both variables assessed: the number of follicles that were PrP sc -positive was detected as well as the intensity of immunostaining in each of them.

Detection of Disease-associated Prion Protein in the Optic Nerve and the Adrenal Gland of Cattle with Bovine Spongiform Encephalopathy by Using Highly Sensitive Immunolabeling Procedures

“…No noticeable differences in the immunolabeling intensity have been found between the two different visualization systems (the avidin-biotin complex system and polymer-based visualization methods such as EnVision+ [Dako]) that are frequently used in laboratories (Monleón et al 2004). Although the TSA system actually improves the signal intensity, this technique requires multiple steps and thus may be unsuitable for routine immunopathological diagnosis.…”

“…This suggests that a highly sensitive immunohistochemical procedure using biotinylated tyramide and including appropriate antigen retrieval is required to detect the presence of minimal quantities of PrP Sc aggregates in tissues (Heggebø, González, et al 2003;Monleón et al 2004;Espenes et al 2006).…”

Distribution of Peripheral PrPSc in Sheep with Naturally Acquired Scrapie

“…For PrP Sc detection by IHC, 3 serial tissue sections (4 µm) spaced 200 µm apart were obtained and assessed as previously described [23]. Briefly, sections were pre-treated with 98% formic acid, proteinase K (4 µg/ml; F. Hoffmann La Roche Ltd, 211 Switzerland) and hydrated autoclaving.…”

Detection of PrPres in Genetically Susceptible Fetuses from Sheep with Natural Scrapie

“…Formalin-fixed samples were trimmed and processed according to standard histopathological procedures. The IHC protocol for PrPres detection was performed as previously described [38]. Briefly, this included immersion in 98% formic acid for 15 min followed by proteinase K (F. Hoffmann La Roche Ltd, Switzerland; 4 µg/ml) treatment for 15 min at 37° and hydrated autoclaving.…”