Optimization of fluorescence staining of zinc and related proteins (ZIP1 and RREB1) in human prostate

Abstract

Abstract Prostate cancer (PCa) is one of the causes of cancer related deaths in men. The current clinical tools for its diagnosis have limitations that makes it unreliable. Thus, there is need for alternative procedures for PCa diagnosis. Several reports have identified zinc as a potential biomarker for PCa diagnosis. The possiblity of using zinc as a biomarker in PCa identification depends on developing a method for detecting intracellular zinc in prostate tissue. This formed the basis of this project. Cellular metabolism of the healthy prostate is altered in prostate malignancy. Studies have confirmed that prostate cancer is characterized by decrease in zinc levels compared to healthy prostate. ZIP1 transporter is responsible for the uptake and accumulation of zinc in healthy prostate epithelial cells and its downregulation has been correlated with decreased zinc levels in malignant prostate tissues. Ras responsive element binding protein 1 (RREB1) is a transcription factor that negatively regulates ZIP1 transporter. RREB1 overexpression was found to correlate with ZIP1 downregulation in prostate malignancy. In this project, intracellular zinc in human PCa tissues were detected using FluoZin-3 zinc indicator dye. The use of FluoZin-3 zinc indicator dye in staining zinc in tissues proved problematic in our experiments. However, by optimizing the fluorescence staining protocol, our results indicate, permeabilizing the tissues before staining with Fluozin-3 zinc dye is an important step. The problem of autofluorescence was encountered in the tissues. The autofluorescence masked specific fluorescence signals and therefore making it difficult to analyze the signals. Sudan Black B and TrueBlack solutions were used to block autofluorescence. Results showed that Sudan Black B reduced autofluorescence but had an impact on the specific fluorescence signals. TrueBlack solution reduced autofluorescence in the tissues. The presence of ZIP1 and RREB1 proteins in the tissues were determined by labeling with specific primary antibodies. The antibodies were optimized for labeling the prostate tissues to determine the antibody titer that will give a high staining intensity and signal-to-noise ratio (SNR).