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Dr Kenneth Wester
Matilda Ahnfelt (technician), Sandra Andersson (technician), Dijana Cerjan (technician), Åsa Edvinsson (technician), Inga Hallin (technician), Linda Oscarsson (technician), Marcus Runeson (technican), Urban Ryberg (technican) and Eva Wahlund (chief technician) and Linda Paavilainen (PhD student).
(i) Handling and storage of antisera, (ii) testing and titration of affinity purified monospecific antibodies, (iii) evaluation and validation of immunostaining test results as well as determination of reliability for immunohistochemistry, (iv) immunostainings on full-scale TMA:s for all approved and validated antibodies, (v) protein extractions for western blot analysis, (vi) western blot analysis of external antibodies.
Affinity purified monospecific antibodies are titrated using a specially designed test-TMA, representing a limited selection of tissues and cells. The titration is done according to a defined schedule where different antibody dilutions and antigen retrieval methods are tested. The working dilution is based on the protein concentration for each antibody. Heat induced epitope retrieval (HIER) in citrate buffer, pH 6 is routinely used for antigen retrieval. When applicable, HIER using high-pH buffer and enzyme based retrieval using proteases are also tested. Microscopical examination is used to evaluate immunoreactivity. Available information from both gene and protein databases as well as in-house produced validation data, i.e., protein arrays and western blots, are considered when assessing the test stainings. For each approved antibody an immunostaining protocol is defined and subsequently applied to the full-scale TMA:s. Automated immunostaining instruments and commercially available detection kits are used to ensure standardization and reproducibility.
Proteins are extracted from in vitro cultured cells and human tissues. Protein extracts are used for validation of all antibodies by Western blot analysis.
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