LAMP2 as a marker of EBV-mediated B lymphocyte transformation in the study of lysosomal storage diseases.

Following the degradative pathway, vesicles loaded with extracellular material, eventually, dock and fuse with lysosomes, acquiring specific membrane markers of these organelles and acid hydrolases responsible for digest their content. The lysosomal-associated membrane protein 2 (LAMP-2), the best characterized lysosomal membrane protein, is found in late stages of endosome maturation and may be used as a marker of lysosome-associated membranes. Lysosomal storage disorders (LSDs) are described by the absence or deficiency in hydrolase activity leading to substrate accumulation within lysosomal components and to the onset of several diseases. It is known that lymphocytes infected by Epstein-Barr virus (EBV) are able to form cytoplasmic vacuoles, which work as a storage compartment for lysosomal acidic hydrolases. At the present study, we validate the EBV as a transforming agent of B lymphocytes in stability studies of long-term stored samples, since the methods used to keep samples in liquid nitrogen and thaw them have all proven to be efficient in samples frozen for up to 2 years. To confirm and investigate some of the most prevalent LSDs in the South of Brazil-Pompe, Fabry and Gaucher diseases-we first measured the enzymatic activity of α-glicosidase, α-galactosidase, and ß-glicosidase in those cytoplasmic-formed vacuoles and then looked to LAMP-2 immunoreactivity by employing confocal microscopy techniques.

An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues.

Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be used. Until now, the efficacy of the processes described depended on the tissue type and on the processing technique, no general recipe for the control of autofluorescence being available. Using paraffin sections of archival formalin-fixed murine liver, kidney and pancreas, we have found that previously described techniques were not able to reduce autofluorescence to levels that allowed direct immunofluorescence labelling. In this work, we aimed at improving currently described methodologies so that they would allow reduction of the autofluorescent background without affecting tissue integrity or direct immunofluorescence labelling. We have found that the combination of short-duration, high-intensity UV irradiation and Sudan Black B was the best approach to reduce autofluorescence in highly vascularised, high lipofuscins' content tissues, such as murine liver and kidney, and poorly vascularised, low lipofuscins' content tissues such as the pancreas. In addition, we herein show that this methodology is highly effective in reducing autofluorescent background to levels that allow detection of specific signals by direct immunofluorescence.