Simultaneous double labelling of routinely processed paraffin tissue sections using combined immunoperoxidase, immunofluorescence, and digital image editing L. Ressel, A. Poli * Department of Animal Pathology, School of Veterinary Medicine, University of Pisa, Viale delle Piagge 2, 56124, Pisa IT, Italy article info Article history: Accepted 7 July 2009 Keywords: Double immunohistological labelling Image editing Immunofluorescence and immunoperoxidase abstract An innovative image editing system based on a sequential immunoperoxidase–immunofluorescence technique on routine histological sections is described. With this technique it is possible to identify dif- ferent antigens in different cells, as well as co-localised antigens in the same cell. The method uses digital image editing to mix two independently captured images into one merged image. The technique was per- formed with indirect immunoperoxidase, followed by sequential indirect immunofluorescence, digital image acquisition and image editing. Multiple staining examples using anti-cytokeratin, anti-vimentin and anti-calbindin antibodies on canine skin and cerebellum, and feline pleural mesothelioma sections were performed in order to investigate the capabilities of the proposed technique. Our data demonstrated that this method can be easily used to assess multiple protein staining studies with minimum laboratory equipment, and that it allows a better structural visualisation of the tissue morphology compared to dou- ble immunofluorescence. Moreover, in contrast to double-immunoperoxidase, with this method it is pos- sible to easily co-localise two different antigens in the same cell compartment. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction A demonstration of multiple antigens in the same formalin- fixed paraffin-embedded tissue section (FFPES) is often desirable for protein localisation or co-localisation studies. Double immun- operoxidase (IP) is widely used (Mason et al., 1983; Vandesande, 1988; Jeurissen et al., 2000; Ramalho et al., 2006) and enables the simultaneous visualisation of different antigens in the same tissue section, thus achieving a good structural view of the speci- men. This technique is time consuming and it is often difficult to discriminate between colours when two antigens are spatially near to each other in the section. Thus this means that it is difficult to detect two co-localised antigens in the same cell (Valnes and Brandtzaeg, 1984; Mason et al., 2000; Borzacchiello and Roperto, 2006). Double or multiple immunofluorescence (IF) have been pro- posed by some authors (Mason et al., 2000; Bombardi et al., 2006; Borzacchiello and Roperto, 2006; Paciello et al., 2007) as quick techniques that can detect different antigens in the same tissue or co-localised proteins in the same cell. However, they do not give a good architectural view of the whole tissue. A few authors have tried to mix the benefits of these two techniques by performing sequential IP and IF staining on the same section and thus obtain- ing two separate pictures (Canese and Bussolati, 1977). Some have tried to get one merged microphotograph in which both signals are detectable using different filters and fluorescent alkaline phospha- tase substrates (Lechago et al., 1979; Ramshaw and Parums, 1992; Tao et al., 1994). Recently Masuda et al. developed a simple double IP on routinely processed paraffin sections using Adobe Ò Photoshop software (Masuda et al., 2008). Here we describe an innovative double indirect immunoperoxidase (IIP) – indirect immunofluorescence (IIF) image processing technique on routinely processed paraffin sections in order to identify different antigens in different cells as well as co-localised antigens in the same cell. The technique uses digital image capture and editing to merge the two independently acquired images from the two different methods (IIP, IIF) of the same microscopic field into one composite image. Since feline mesothelioma cells express both cytokeratin and vimentin (Bacci et al., 2006), we used this neoplastic tissue to investigate the possibility of the simultaneous visualization of these two antigens in the same cell. 2. Materials and methods 2.1. Specimens and antibodies Formalin fixed paraffin-embedded tissue samples from canine normal skin (n = 3) and cerebellum (n = 2) were selected from the archives of the department of Animal Pathology at the University of Pisa. A case of feline pleural mesothelioma was also investigated. For each block, four lm thick sections were cut and mounted on 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.07.003 * Corresponding author. Tel.: +39 050 2216982; fax: +39 050 2216914. E-mail addresses: lorenzo.ressel@poste.it (L. Ressel), apoli@vet.unipi.it (A. Poli). Research in Veterinary Science 88 (2010) 122–126 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc