Pilot testing of a simple alternative tissue processing method for immunofluorescence staining

Abstract

Background: The image quality of tissues obtained with high-resolution imaging systems depends on several factors including the quality of sections prepared by different tissue processing methods. Fresh frozen sections and formalin-fixed paraffinembedded (FFPE) sections are the two routinely employed methods in histopathological examination having their own merits and demerits. Aims and Objectives: We tested the feasibility of a hybrid tissue processing method that could reduce the processing time without compromising the quality of processed and stained tissues. Materials and Methods: Bovine pancreatic tissue was utilized to standardize the process. The steps included fixation of tissue in formalin, followed by embedding in optimal cutting temperature compound and then cryo-sectioning to obtain 50 μm slices for immunostaining. The prepared thick slices were immunostained with islet markers and assessed by confocal imaging. Results: The images revealed that the staining quality was comparable with FFPE sections, though the tissue processing time was significantly curtailed. The modified Leica microsystems scoring system revealed a nearly 80% overall total score for the processed tissue specimens, indicating acceptable levels of quality of the tissues. We also observed that the staining quality of these formalin-fixed cryo-slices was preserved without tissue deterioration for up to 1 month when stored in formalin, indicating its storage potential for future applications. Conclusion: The proof of concept of the hybrid tissue processing method tested in this study will encourage researchers to extrapolate its use to the human pancreas, other solid organs, and other staining techniques for evaluating its general utility.