Abstract
The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.
Highlights
Immunohistochemistry (IHC) is an important technique for pathology diagnostics and investigative studies[1]
Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin
antigen retrieval (AR) done in the steam cooker showed the best results when compared with AR done in the microwave oven or immunohistochemistry without AR
Summary
Immunohistochemistry (IHC) is an important technique for pathology diagnostics and investigative studies[1]. During the AR step, the tissues are subjected to high temperatures in a solution containing metallic salts (sulfate zinc, zinc chloride and aluminum chloride) dissolved in a sodium citrate buffer at pH 6.0-9.0 or distilled water[5]. This procedure is very simple, it contains factors that can affect immunostaining, such as heating conditions and the pH of the solutions[6]. We evaluated two heating methods for AR-IHC, the microwave oven and the steam cooker, in order to discover which method of immunostaining was more efficient, and to standardize this methodology for the invertebrate nervous system
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