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Fixed cells were permeabilized by 0.5% Triton X-100 (BioShop, Canada) in PBS for 15 min, blocked for 45 min in 3% BSA (BioShop, Canada) + 0.1% Tween-20 (BioShop, Canada) in PBS, and then incubated overnight at 4 °C with primary antibodies diluted in 5% normal donkey serum (NDS, Jackson Immunoresearch, UK)

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This involves doctors performing a series of evaluations, laboratory tests, and specialized imaging examinations