Abstract
Two important criteria used to select an antibody diluent for robust immunohistochemistry are: (1) optimal signal/noise ratio, and (2) long-term stability when diluted. For immunohistochemistry with Fanconi anemia, complementation group D2 (FANCD2) in formalin-fixed-paraffin-embedded breast cancer tissue, eight experimental and eight commercial diluents were evaluated. Staining intensity was quantitated with Aperio digital image analysis software. An H-Score was calculated for FANCD2 and isotype control staining for each diluent. Signal/noise ratio was defined as H-score of the antibody divided by H-score of the isotype control. The ratios ranged from 0.46 to 135.9 for different diluents. Casein in Tris-based buffer produced the greatest signal/noise ratio. The two best commercial and four best experimental diluents were selected for evaluation of the diluted FANCD2 antibody stability at 4°C, room temperature, and 37°C, respectively. Three diluents were eliminated from further investigation after 2 weeks because of suboptimal staining. Evaluation of room temperature and 37°C storage ended at 1 month, and evaluation of 4°C storage ended at 6 months. FANCD2 antibody was stable for at least 1 month at room temperature and 37°C, and for at least 6 months at 4°C in casein-based diluents, as well as the diluent containing bovine serum albumin and normal horse serum.
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