Grace Bio-Labs has developed a method to cast polymetric nitrocellulose onto glass microscope slides to make a film-based product designed specifically for tissue processing and cytochemistry. The chemical bond linking film to glass is stable to temperature/solvent/pH conditions of all cytochemical assays including PCR and is autoclavable. A combination of nitrocellulose polymer and proprietary chemistry is resistant to standard Reblot™ and antigen retrieval protocols, while maximizing protein binding and stability. The film surface is flat with round pores approximately 0.1um diameter and is amenable to automated printing with contact or non-contact devices. Films are less than 20um thick with an open internal pore structure and are compatible with virtually all detection systems, including fluorescent, chemiluminescent, radiographic and colorimetric. In addition, films have negligible autofluorescence and can be cleared to transparency for high resolution imaging.
ONCYTE® film slides are manufactured to the highest standards and quality tested, it provides excellent reliability and reproducibility. There are many existing stock film patterns available, and Grace has the ability to customize film patterns to the needs of their customers. In addition, Grace offers strong R&D support for new applications and quality support for variance control of key performance indicators.
ONCYTE® film slides are compatible with a variety of counterstains including Mayer’s hematoxylin, nuclear fast red and light green SF. Gill’s hematoxylin is recommended (0.05% HCl may be used to de-stain). Eosin (0.5% diluted in isopropanol). In some cases counterstains may bind to the film, this usually does not interfere with the microscopic examination of specimens. Film slides are made transparent for microscopic and imaging applications by xylenes or immersion oil with a refractive index of 1.515. For aqueous mounting Vectashield (Vector Labs), slow Fade and Flow Fade Light (Thermo Fisher Scientific) are recommended.
The film surface is flat with round pores approximately 0.1um diameter. Films are less than 20um thick, and bound to a standard 25mm x 75 mm glass (or plastic) microscope slide. The center-to-center spacing of ONCYTE® multi-films matches that of 96 and 384 well microtiter plate format. Multi-films can be processed using the Grace Bio-Labs ProPlate® multiarray system.
- Ideally suited for use in most cytochemistry assay protocols including ICC, ISH, ISPCR and HTS
- Optimal for infectious disease immunogen discovery
- Film slides provide superior specimen adherence, outperforming silane and protein treated glass slides
- Increases sensitivity of frozen section immunocytochemistry and for printing soft tissues
- Specimens may be applied to a film slide by any conventional method including centrifugation, cryostat mounting, tissue printing pipetting, and array depositors
- Specimens may be chemically fixed using aqueous fixatives or compatible alcohols
- Soluble analyte may be bound to film slides by air drying, UV crosslinking, baking or microwaving
- Slides are chemically resistant to reagents typically used in most assays for cell analysis including 50% formamide
- Film slides are not recommended for use with acetone, ethanol and methanol. Isopropanol and butanol are common substitutes
Henkel, Sebastian, Robert Wellhausen, Dirk Woitalla, Katrin Marcus, and Caroline May. "Epitope Mapping Using Peptide Microarray in Autoantibody Profiling." Methods in Molecular Biology (Clifton, N.J.)1368 (2016): 209-24.
Yentrapalli, Ramesh, Omid Azimzadeh, Anne Kraemer, Katharina Malinowsky, Hakan Sarioglu, Karl-Friedrich Becker, Michael J. Atkinson, Simone Moertl, and Soile Tapio. "Quantitative and Integrated Proteome and MicroRNA Analysis of Endothelial Replicative Senescence." Journal of Proteomics 126 (2015): 12-23.
Wolfe, Adam, R. Debeb, Bisrat Lacerda, G. Larson, Lara Bambhroliya, Richard Huang, Arvind Bertucci, Xuelin Finetti, Francois Birnbaum, Pascal Laere, Daniel Diagaradjan, Steven Ruffell, Parmeswaran Trenton, Brian Chu, Nicholaus Hittelman, J. Diehl, Khoi Levental, Walter Ueno, and Michael Woodward. "Simvastatin Prevents Triple-negative Breast Cancer Metastasis in Pre-clinical Models through Regulation of FOXO3a." Breast Cancer Research and Treatment 154, no. 3 (2015): 495-508.
Which concentrations of antibodies or proteins should be printed on the ONCYTE® Film Slides? For purified proteins a concentration of about 0.05-1 mg/ml is optimal for most applications, the upper end of this range is recommended for antibodies.
Should printing be performed at low temperatures? No, printing may be performed at room temperature as long as humidity is maintained at around 50-55% to avoid sample evaporation during the process.
My protein sample contains urea, is this going to affect protein binding to ONCYTE® Film Slides? No. Samples containing urea are routinely printed on ONCYTE® Film Slides. Slides are chemically resistant to reagents typically used in most assays for cell analysis including 50% formamide. Film slides are not recommended for use with acetone, ethanol and methanol. Isopropanol and butanol are common substitutes. DMSO could negatively affect nitrocellulose too, therefore its concentration should not exceed 5%.
Can traditional western blot blocking buffers be used for blocking protein microarrays? The composition of the blocking buffer should be optimized for each experimental setting. Colorimetric and chemiluminescent detection are compatible with the blocking buffers commonly used for western blot, such as PBS or TBS containing 1-5% non-fat milk. Fluorescent detection, on the other hand, can be hampered by background fluorescence typically associated with protein–based blocking buffers. Super G Protein array blocking buffer has been optimized for fluorescent assays and produces a very high signal to noise ratio, minimizing the background fluorescence.
How do I image my microarray results? ONCYTE® Film Slides are compatible virtually with all detection systems: isotopic, chemiluminescent, chromogenic, and fluorescent detection are all viable options.
How should printed ONCYTE® Film Slides be stored? Non-printed slides should be stored at room temperature in their original packaging. Printed slides can be stores at 4°C for short-term storage or at -20°C for long-term storage. An overnight incubation at 4°C is recommended after printing in order to maximize protein binding before use.
What kind of controls should be included in the microarray analysis? IgG pre-labeled with a fluorophore should be spotted on the array to check for proper protein binding. Buffer-only spots can be used for background subtraction, as well as secondary antibody-only stained film slides. Replicates should also be spotted on the array for each protein.
Can ONCYTE® Film Slides be ‘stripped’ and re-used? No. It is not advisable to re-use ONCYTE® Film Slides.
ONCYTE® Guide to Protein Microarrays This 24-page document provides detailed methods for creating and developing protein microarrays using ONCYTE® porous Nitrocellulose Film Slides. Sections of this manual also include a troubleshooting guide and other helpful references.