title: Immunohistochemical double-staining of renal allograft tissue: critical assessment of three different protocols.
abstract: Quantitative analyses of renal allograft tissue using immunohistochemical double-staining could be a useful tool to extend the existing knowledge on renal allograft immunopathology. Due to technical reasons, this method has been only rarely applied in the past. The use of indirect immunohistochemistry for double-staining bears the risk of nonspecific cross reactions between the two staining sequences. To date, various procedures have been refined to avoid such cross reactions. Here we assessed the validity of three different protocols for indirect immunohistochemical double-staining on frozen sections of renal transplant biopsies ( n=12). Both colocalized antigens and antigens with a non-overlapping distribution were stained according to each of the three protocols. Differentiation between the two staining sequences was achieved by employing different colored substrates of alkaline phosphatase (protocol 1), different enzymes (peroxidase and alkaline phosphatase) together with the use of 3,3'-diaminobenzidine-tetrahydrochloride substrate in the first staining sequence (protocol 2), or primary antibodies from different species (protocol 3). Sensitivity and specificity of each protocol were determined by quantitative comparison with control single-stainings of adjacent sections. Sensitivity of the first staining sequence was about 100% with each of the three protocols investigated. In the second staining sequence, sensitivities of protocols 1 (50%) and 2 (54-66%) were much lower than of protocol 3 (100%). Specificity of the second staining sequence was only 44% with protocol 1 compared with 98% with protocol 2 and 100% with protocol 3. In conclusion, protocols 1 and 2 are not recommended for quantitative double-staining analyses. In contrast, protocol 3 provided maximum sensitivity and specificity, even for antigens that are colocalized on the same cell type. Thus, the use of primary antibodies from different species is by far the most reliable technique for quantitative double-staining analyses in renal allograft tissue.
authors.affiliation: Department of Internal Medicine-Neophrology, University Hospital Charité, Campus Mitte, Humboldt-University Schumannstrasse 20/21, 10117 Berlin, Germany. johannes.waiser@charite.de
authors.forename: Johannes
authors.initials: J
authors.lastname: Waiser
authors.forename: Stefan
authors.initials: S
authors.lastname: Schwaar
authors.forename: Torsten
authors.initials: T
authors.lastname: Böhler
authors.forename: Birgit
authors.initials: B
authors.lastname: Rudolph
authors.forename: Kerstin
authors.initials: K
authors.lastname: Dell
authors.forename: Klemens
authors.initials: K
authors.lastname: Budde
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
authors.forename: Hans-Hellmut
authors.initials: HH
authors.lastname: Neumayer
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D001707:Biopsy, Needle; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016030:Kidney Transplantation; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D014184:Transplantation, Homologous / Q000276:immunology / Q000473:pathology*
chemical_list: D015703:Antigens, CD