Ana Cathia Magalhães1, Claudio Rivera2. 1. Neuroscience Center, University of Helsinki, Viikinkaari 4, 00014 Helsinki, Finland. Electronic address: Ana.c.magalhaes@helsinki.fi. 2. Neuroscience Center, University of Helsinki, Viikinkaari 4, 00014 Helsinki, Finland; Aix-Marseille University, UMR S901, Parc Scientifique de Luminy, 13009 Marseille, France; INSERM, INMED, Parc Scientifique de Luminy, 13009 Marseille, France. Electronic address: Claudio.rivera@helsinki.fi.
Abstract
BACKGROUND: Immunohistochemistry (IHC) studies in paraffin-embedded sections can be challenging due to epitope masking. To counteract this problem the microwave oven is widely used for heat-induced epitope retrieval (HIER). However, with this method important parameters cannot be specifically controlled, such as the intensity and duration of heating. NEW METHOD: We describe here a consistent and sensitive HIER method that uses a device for epitope retrieval in tissue sections, the decloaking chamber NxGen. With this method, heat temperature and time can be accurately set. Both qualitative (sensitivity and specificity of positive immunostaining) and quantitative (amount of positive-stained cells) analyses were compared between the microwave oven approach and the decloaking chamber, in paraffin-embedded sections from embryonic mouse brain. RESULTS: We found that the decloaking chamber-based HIER method was preferable to the commonly used microwave oven for the immunodetection and discrimination in mouse brain sections of single Olig2-positive cells, a marker of oligodendrocyte precursor cells. Both automated (ICY software) and manual counting (Adobe Photoshop software) showed a significant underestimation of Olig2-positive cells in microwave oven-treated sections compared to decloaking chamber-treatment. COMPARISON WITH EXISTING METHODS: Compared to other IHC procedures for cell automated quantification, the presently established protocol is easy to use, fast, and effective for the immunodetection and quantification of Olig2 in the developing mouse telencephalon. CONCLUSIONS: We conclude that the combination of decloaking chamber-based HIER method and spot detector in ICY software is a reliable and valuable tool, suited to basic research and clinical studies.
BACKGROUND: Immunohistochemistry (IHC) studies in paraffin-embedded sections can be challenging due to epitope masking. To counteract this problem the microwave oven is widely used for heat-induced epitope retrieval (HIER). However, with this method important parameters cannot be specifically controlled, such as the intensity and duration of heating. NEW METHOD: We describe here a consistent and sensitive HIER method that uses a device for epitope retrieval in tissue sections, the decloaking chamber NxGen. With this method, heat temperature and time can be accurately set. Both qualitative (sensitivity and specificity of positive immunostaining) and quantitative (amount of positive-stained cells) analyses were compared between the microwave oven approach and the decloaking chamber, in paraffin-embedded sections from embryonic mouse brain. RESULTS: We found that the decloaking chamber-based HIER method was preferable to the commonly used microwave oven for the immunodetection and discrimination in mouse brain sections of single Olig2-positive cells, a marker of oligodendrocyte precursor cells. Both automated (ICY software) and manual counting (Adobe Photoshop software) showed a significant underestimation of Olig2-positive cells in microwave oven-treated sections compared to decloaking chamber-treatment. COMPARISON WITH EXISTING METHODS: Compared to other IHC procedures for cell automated quantification, the presently established protocol is easy to use, fast, and effective for the immunodetection and quantification of Olig2 in the developing mouse telencephalon. CONCLUSIONS: We conclude that the combination of decloaking chamber-based HIER method and spot detector in ICY software is a reliable and valuable tool, suited to basic research and clinical studies.