Optimisation of protocols for detection of free cholesterol and Niemann-Pick type C 1 and 2 protein

Abstract: The purpose of this project was to optimise the protocols for detection of free cholesterol and NPC 1 and NPC 2 proteins. Paraffin embedded human and rat tissues, cellblocks and cytospins of HepG2 and HeLa cells were used for immunohistochemistry to try out the best antibody dilutions and unmasking method of the antigen. Adrenal tissue was used to stain lipids with Filipin. The dilution that worked best for the NPC 1 was 1:150 and with EDTA unmasking. For the NPC 2 the dilution 1:100 was optimal and with Citrate as unmasking method. NPC 1 was highly expressed in ovary tissue, stomach epithelium, HeLa cells and rat kidney and liver, while NPC 2 was highly expressed in neurons and astrocytes in Alzheimer’s disease, seminiferous tubules in testis, neurons in intestine, neurons in healthy brain tissue and HeLa cells. The cholesterol inducing chemical U18666A was applied to HepG2 cells but no alteration in lipid staining was observed and NPC protein expression was similar at all doses applied. Filipin staining worked well with a concentration of 250µg/mL and Propidium Iodide with concentration 1mg/mL for nuclei stain was optimised at 1:1000.The fixation of cells before lipid stain and immunoperoxidase staining has to be evaluated further as the fixations used, 10% formalin and acetone, had adverse effects on the antigen.        In this project methods were optimized for lipid and NPC protein staining for further application in disease investigations.