J. Comp. Path. 2007, Vol. 136, 273^278 SHORT PAPER Cytokine Expression in Paraffin Wax-embedded Tissues from Conventional Calves M. Pedrera, P. J. Sa ´nchez-Cordo ´n, J. L. Romero-Trevejo, A. I. Raya, A. Nu ´n ˜ez * and J. C. Go ´mez-Villamandos Departamento de Anatom|¤ a yAnatom|¤ a Patolo L gica Comparadas, Facultad deVeterinaria, Universidad de Co L rdoba, Edi¢cio de Sanidad Animal, Campus de Rabanales, 14014, Co L rdoba, Spain, and * Pathology Department, Veterinary Laboratories Agency,Weybrigde,Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK Summary The cross-reactivity of antibodies against human tumour necrosis factor (TNF)a , interleukin (IL)-1a , IL-1b and porcine IL-6, and the distribution of immunolabelled cells were evaluated on para⁄n wax-embedded tissues from ¢ve healthy calves.The tissues were ¢xed in10% bu¡ered formalin or Bouin’s solution and processed for structural studies and immunohistochemical studies by the avidin^biotin^peroxidase technique. Bouin 0 s solution proved to be the more suitable ¢xative and Tween 20 the most e¡ective antigen unmasking technique for increasing detect- able antigenicity. Constitutive expression of TNFa , IL-1a , IL-1b and IL-6 by di¡erent cell populations, mainly macrophage-like cells, was detected. Lymphoid organs displayed a higher presence of immunolabelled cells than did lung, liver or kidney.TNFaand IL-1a appeared as the predominant cytokines, especially in the gut-associated lymphoid tissue of the ileum and in the regional mesenteric lymph nodes.The results will facilitate investigation of the role of these cytokine-producing cells in in£ammatory disease processes in calves. r 2007 Elsevier Ltd. All rights reserved. Keywords: cattle; cytokines; immunohistochemical technique The development and production of new monoclonal antibodies for immunohistochemistry against species- speci¢c cytokines is both time-consuming and expen- sive. For this reason, it is desirable to investigate whether existing anti-cytokine antibodies cross-react between di¡erent animal species (Pedersen et al ., 2002 ). Despite possible loss of antigenicity, tissue samples subjected to ¢xation and para⁄n wax embedding are preferable to frozen sections in respect of ease of hand- ling and preservation of tissue morphology (Gonza Ł lez et al ., 2001 ; Tingstedt et al ., 2003 ). Moreover, the use of antigen retrieval steps facilitates the unmasking of anti- gens and epitopes and overcomes the antigenicity de- crease caused by cross-linking during formalin ¢xation (Miller et al .,1995 ; Ramos-Vara, 2005 ). Few studies have used immunohistochemistry on wax-embedded bovine tissue samples to elucidate in- situ expression of cytokines in disease. Furthermore, the baseline immunohistochemical expression of proin- £ammatory cytokines in bovine para⁄n wax-em- bedded tissues has not been established. The aims of this immunohistochemical study with a panel of commercially available human and porcine antibodies against tumour necrosis factor (TNF)a , in- terleukin (IL)-1a , IL-1b and IL-6 were to evaluate antibody cross-reactivity and to describe the tissue dis- tribution of these cytokines in healthy calves for future comparisons with diseased calves. Five apparently healthy, conventionally reared Frie- sian calves (three male, two female), aged 3 months, were obtained from farms free of tuberculosis, brucel- losis, bovine leukosis virus, bovine viral diarrhoea and infectious bovine rhinotracheitis. Their live weight was www.elsevier.com/locate/jcpa ARTICLE IN PRESS 0021-9975/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi: 10.1016/j.jcpa.2007.02.002 Correspondence to: J.C. Go Ł mez-Villamandos (e-mail: jcgomez@uco.es).