Microwave-assisted immunostaining: a new approach yields fast and consistent results.

Advances in microwave technology permitted the development of new antigen labeling techniques. The recent microwave development of a true variable wattage unit designed for laboratory use and an apparatus for dampening standing wave radiation patterns have allowed investigators to better control the conditions within a microwave cavity. Thus, operating limits thought to be endemic to microwave-assisted protocols could be effectively mitigated. Standard protocols for histochemistry call for prolonged incubations and numerous rinses that add considerable time to the procedure. Here, we present microwave-assisted staining protocols for floating rat brain sections and cultured rat hippocampal cells. Acetylcholinesterase (ACHE) histochemistry and immunocytochemistry were conducted inside a specially designed and configured laboratory microwave oven. As a control additional tissue sections were stained on the bench and treated in the same manner as those in the microwave. Labeling was minimal in the control tissue, but specific, high contrast staining was present in the microwave group. Tissues were evenly stained with minimal background, and anatomical structures were easily detected. Also, the differences between lesioned and intact sides of the brain were obvious and agreed with previous observations. Microwave-assisted methods resulted in significantly shorter protocol times (approximately 10-fold) resulting in staining patterns of equal or superior quality to those obtained using conventional methods.

The use of mouse models of breast cancer and quantitative image analysis to evaluate hormone receptor antigenicity after microwave-assisted formalin fixation.

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.

Advanced laboratory techniques for sample processing and immunolabeling using microwave radiation.

A better understanding of improved microwave technology has increased the benefits and versatility of the technique as it applies to all aspects of immunohistochemistry. The role of continuous magnetron power output (wattage) combined with precise control of sample heating demonstrated their significance to complex labeling protocols. Here, we present results for microwave-assisted formaldehyde fixation and its effect on GFP expression in transfected HeLa cells. Rat brain sections and cultured hippocampal cells were labeled with 11 different primary antibodies using a unified microwave protocol. Microwave-assisted immunohistochemistry made it possible to sequentially label tissues and cells with several primary antibodies in a very short period of time with excellent labeling characteristics.

Microwave and digital imaging technology reduce turnaround times for diagnostic electron microscopy.

The contributions of microwave methods and digital imaging techniques, when taken together, can reduce routine specimen processing and evaluation for diagnostic electron microscopy to a time frame never thought possible. Significant improvements in both technologies over the last 5 years led the authors to evaluate their combined attributes as the most likely candidate to provide a realistic solution in the reduction of turnaround times for diagnostic electron microscopy. For diagnostic electron microscopy to compete favorably with immunohistochemistry and other ancillary diagnostic techniques, it must improve its turnaround time. To evaluate this hypothesis the microwave-assisted processing results of over 2,000 diagnostic cases were evaluated as was a digital image administration system used for the acquisition and dissemination of diagnostic results. The incorporation of both technologies resulted in turnaround times being reduced to 4 h or less.