Designing and Validating a New Method the TUNEL Microwave (TUNEL-MW) for Rapid Quantification of Apoptosis in Islets Following Isolation and Post-Thaw

Review Data

This work is based on application of a microwave oven system to TUNEL, according to a technical innovative approach. Assays have been conducted on different tissue sections using either TUNEL alone, or TUNEL upon staining with H&E to obviate problems associated with markers and dyes.

Microwave oven use was already validated in immunocytochemistry, since it helps diffusion of reagents into tissue specimens, promoting fixation and homogeneous heat diffusion in the tissues, with no morphological changes. Energy generated by microwave ovens breaks covalent bonds and hydrogen links.

The most advantageous feature proposed by this method is cutting off specimens process time and costs. Performance of the method also is based on type of the starting tissue specimen. For instance pancreatic tissue, in its whole, may be auto fluorescent, which may interfere with fluorescent staining, thereby compromising quality of the results. As for pancreatic tissue, autofluorescence may adversely affect fixation and staining possibly due to picric acid contained in the Bouin’s reagent for tissue fixation.

Here specimens’ analysis was conducted only on isolated islets of Langerhans (thereafter prepared for TUNEL). While the tissue here is comprised of isolated islets only, procedures for fixation (choice of reagents important) should be standardized as homogeneously as possible to avoid pending issues. Anyway, here the proposed fixed experimental parameters, aimed to prevent false-positive results, reinforce impact of the proposed procedure.

This paper is written quite clearly, in its sections, with correct and significant statistics.

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