We previously observed decreased p53 immunostaining over time in paraffin-embedded sections of ductal carcinoma in situ of the breast of women; these sections had been stored on slides at room temperature. This observation suggests that slide storage adversely affects p53-immuno-staining intensity and could result in spurious negative staining for p53 in patient samples. The goals of this study were to determine the time course and factors influencing loss of p53 immunoreactivity and to investigate whether a similar loss of reactivity occurs with other antigens commonly used to study breast cancer. Serial sections cut from 12 formalin-fixed, paraffin-embedded, p53-positive invasive ductal carcinomas of the breast were stored on slides at room temperature or at 4 °C, with or without an additional paraffin coating, for 2, 4, 8, or 12 weeks. For each case, freshly cut slides from the same block (day 0) and stored slides were simultaneously stained for p53 by use of an automated immunostainer. Slides cut from formalin-fixed, paraffin-embedded breast carcinomas and stored for 12 weeks were also stained for factor VIII-related antigen (n = 12), estrogen receptor (ER) (n = 9), and Bcl-2 protein (n = 9). The staining intensity of all slides was assessed by visual microscopic examination and was also quantified by image analysis. Quantitative results were expressed as a percentage (mean ± standard error) of the staining intensity on day 0. Data were analyzed by the Friedman Repeated Measures Analysis of Variance on Ranks, with statistical significance set at two-sided P <.05. The intensity of p53 staining decreased over time in nine (75%) of the 12 cases studied. In three (or 25% of all cases studied) of the nine cases that showed decreased p53 staining, slides stored for 12 weeks were scored as p53 negative. Antigen loss on slides stored at 4 °C was significantly less than that on slides stored at room temperature at all time points (all P <.05). At 12 weeks, the average staining intensity of slides stored at 4 °C was 33.2% ± 9% of that on day 0 compared with 8.4% ± 3% of that on day 0 for slides stored at room temperature ( P <.001). Paraffin coating of the sections did not significantly diminish antigen loss at either room temperature or 4 °C, except for slides stored at room temperature for 12 weeks. The intensity of factor VIII staining decreased in nine of 12 cases (average staining intensity, 37.3% ± 6% of that on day 0 at 12 weeks; P =.0001). The intensity of ER and Bcl-2 staining decreased in all nine cases studied at 12 weeks (average staining intensity, 14.0% ± 6% and 21.0% ± 4% of that on day 0, respectively; P =.0001 for each). Slide storage, particularly at room temperature, results in substantial loss of p53 reactivity, with some p53-positive cases becoming p53 negative after 12 weeks of storage. Substantial loss of immunoreactivity for factor VIII, ER, and Bcl-2 occurs on slides stored at room temperature for 12 weeks. Storage of unstained slides for up to 12 weeks may lead to false-negative immunostaining for p53 and other antigens. [J Natl Cancer Inst 1996; 88: 1054–9]