The results are shown in Figure 4B. global views of protein amounts and activation status.4,5 These high-throughput data sets are then the basis for a variety of new and innovative bioinformatics tools that provide unique insights into biological states and disease processes.6,7 However, the study of histological sections presents a number of challenges compared with other sample types. In particular, it is difficult to maintain the two-dimensional histopathological information present within a sample while also performing multiplex molecular analysis. To some degree, the development of laser-based microdissection tools has solved this problem.8 However, microdissection studies are tedious and time-consuming, frequently requiring the user to pool together the procured cells and thus drop important geographic information of the cell populations and subpopulations that are captured. Thus, there is a need in the molecular pathology community for analysis methodologies that combine two-dimensional histopathology with multiplex arrays.9 Indirect layered peptide array (iLPA) technology is one such example. In the present study, we describe a prototype version of iLPA and assess its capability for high-throughput tissue section measurements. Materials and Methods Tissue Samples Prostatectomy cases were obtained from the National Institutes of Health and the National Naval Medical Center under an Institutional Review Board-approved protocol. Whole-mount prostate cancer cases were ethanol-fixed and paraffin-embedded as described previously.10 Tissue sections were cut to 5- to 10-m thickness for the iLPA protocol. Labial minor salivary gland tissues were obtained from nine patients with primary Sj?grens syndrome and two healthy volunteers and were acquired and used in accordance with approvals from the National Institute of Dental and Craniofacial Research human subject review committee. Immediately after removal, specimens were placed in OCT compound (Miles, Elkhart, IN), snap-frozen in methyl butane on dry ice, held overnight at ?70C, and then stored in liquid nitrogen until use. Tissue samples were cut at a 10-m thickness for the iLPA protocol. Each section was placed on a charged glass slide. Tissue microarray (TMA) slides were obtained from the Tissue Array Research Program (TARP) lab at the National Cancer Institute (the individual level of gray of each object. TAK-901 The data were then imported to Microsoft Excel 2000 (Microsoft, Seattle, WA) and saved as a spreadsheet. Enhancement of images was performed in the ImagePro 4.5 program using the automatic feature best-fit and is presented as an enhanced image. The data from Microsoft Excel were imported to PartekPro 6.2. (Partek Inc., St. Charles, MO). Principal component analysis, group profile, analysis of variance statistics, and SD modules of the PartekPro software package were used to analyze the results. Results and Discussion The iLPA platform is usually shown schematically in Physique 1. Membranes were coated with peptides specific to antigens of interest, and tissue sections were then incubated with a cocktail of antibodies against target proteins. After washing, the antibodies were released from the section and exceeded through the analysis layers while maintaining their two-dimensional position. The antibodies were specifically captured by their target peptides and subsequently detected using standard secondary antibody-based methods. Open in a separate window Physique 1 Schematic of the iLPA system. Each membrane is usually coated with a different peptide or antigen specific for an antibody of TAK-901 interest. An antibody set is usually applied to a tissue section and subsequently captured and analyzed by the appropriate membrane, while maintaining the two-dimensional information present in the sample. The protocol for releasing the antibody from the tissue section was decided empirically, starting with traditional column-based methods for antibody purification. The effect of different buffers, salt concentrations, and pH conditions on both the antibody detachment step and the subsequent antibody re-binding step to the membrane Rabbit Polyclonal to Akt was decided. Ultimately, release and re-binding of antibodies was optimal using sodium phosphate buffer, pH 7, and subjecting the histology slide and membranes to heat and capillary fluid flow as described in Materials and Methods. This protocol effectively released the majority of bound antibody from the tissue section and permitted efficient re-binding of the antibody to its appropriate peptide-coated membrane. Physique 2 demonstrates analysis of prostate-specific antigen (PSA) in a whole-mount prostate sample in a clinical specimen from a patient with TAK-901 cancer. Physique 2A shows a low-power, annotated image of the hematoxylin and eosin-stained histology slide, indicating the location of normal epithelium, epithelial.