Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides

Abstract

Storage of tissue slides has been claimed to induce dramatically reduced antigen detection particularly for immunohistochemistry (IHC). With tissue microarrays, the necessity to serially cut blocks in order to obtain as much material as possible is obvious. The presumed adverse effect of storage might hamper such an approach. The authors designed an experimental setting consisting of four different storage conditions with storage time of tissue slides of up to 1 year. Detection of proteins, DNA, and mRNA was performed using IHC and in situ hybridization techniques. Slight but significant changes in IHC occurred over time. The most important factor is the primary antibody used: four showed no significant changes, whereas limited decreases in 8 antibodies could be detected by image analysis. Whether the antigen was nuclear or cytoplasmic/membranous did not matter. No major differences between different storage conditions could be shown, but storage at 4C was overall the best procedure. Furthermore, gene copy number aberrations, chromosomal translocations, and the presence of mRNA could be detected on slides stored up to 1 year. In conclusion, in tissues optimally formalin fixed and using modern histological techniques, only minute changes in tissue antigenicity are induced by long-term storage.

Figure 1.

The picture segmentation demonstrated at cylinder stained for p53 at baseline. Background and counterstaining is blue. Strong positive staining is red (0-100 gray levels) and regarded as true positive. Orange (101-175) and yellow (176-220) were considered negative, mainly constituting cytoplasmic background staining. A-B, bar = 1.0 mm.

Figure 2.

Figure 2 shows the results of the ANOVA analyses for each antibody (row) and storage condition (column). The color of each box indicates the level of significance in the ANOVA analysis of the whole time series of observations (baseline, 1 week, and 1, 3, 6 and 12 months). No statistical difference over time is thus indicated by a green box. The numbers within the box indicate the difference in gray value (n=256 gray levels) concerning the maximal difference at any time point (left) and the average of all observations (right) versus the baseline value. 4 = 4C, pf = paraffin-coating, RT = room temperature.

Figure 3.

The series of pictures show immunohistochemical staining results of the same cylinder at baseline (A) and after 12 months of storage at RT (B), at RT with a coating of paraffin (C), at 4C (D), and at 4C with paraffin coating (E). The graph shows the overall mean gray value for the different storing times at different storing conditions. Diaminobenzidine was used as chromogen showing brown color. 4 = 4C, pf = paraffin-coating, RT = room temperature. A-E, bar 100 µm.

Figure 4.

mRNA detection of κ light chain in freshly cut tonsil tissue (A) versus slides stored for 12 months at RT without a paraffin coating (B) and stored for 12 months at 4C with a paraffin coating (C). A-C, bar 0.1 mm.