Trypan blue staining method for quenching the autofluorescence of RPE cells for improving protein expression analysis

Abstract

Abstract Purpose The purpose of this study is to develop and validate Flow Cytometry (FC) and Immunohistochemistry (IHC) methods for rapid and accurate measurements of cell proteins corresponding specific fluorescence signals above background noise using Trypan blue (TB) for quenching the autofluorescence (AF) emitted by Retinal pigment epithelial (RPE) and Ciliary body stem (CBSC)cells. Methods RPE and CBSC cells were isolated, cultured and trypsinized using already published methods. AntiRPE65 immunolabelled cells were post treated with 10, 20 and 40 µg/ml TB at 4C for 10 min for FC analysis. 3‐5 mm small pieces of retinal tissue were pre treated with 20, 200 and 2000 µg/ml of TB at room temperature for 15 min, followed by embedding in paraffin wax, cutting into 3 µm thick retinal tissue sections (RTSs), dewaxing, anti RPE65 immunolabelling and observing under a microscope.Immunolabelling with isotype‐matched unspecific Abs, primary and secondary antibody were used as controls. Cytomicx RXP and Adope photoshop were used for FC and IHC results analysis. Results FC and IHC results showed that post‐treatment of immunolabelled RPE cells and pre‐treatment of retinal tissue pieces with 20 µg/ml of TB reduce the AF and facilitate to detect the fluorescence signals corresponding to specific cell proteins with higher mean fluorescence intensity (MFI). Conclusion We concluded: 1.‐Incubation of cells and tissues with 20 µg/ml of TB reduces AF. 2.‐For FC analysis, the cells should be post‐treated with TB and for IHC analysis, the tissues should be pre‐treated with TB. 3.‐The methods significantly increase the quality and value of cell protein analysis performed by FC and IHC techniques.