Novel contrasting and labeling procedures for correlative microscopy of thawed cryosections.

摘要: One of the major challenges for correlative microscopy is preparation sample; protocols transmission electron (TEM) and fluorescence (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure Tokuyasu sections that yields both excellent positive membrane contrast in TEM bright probe labeled on section. 2+Staining involves immunolabeled with 1% osmium tetroxide, 2% uranyl acetate lead citrate sequential steps, followed by embedding 1.8% methyl cellulose. In addition, demonstrate amplification fluorescent signal introducing additional antibody incubation steps immunolabeling procedure. The methods were validated using integrated laser microscope (iLEM), a novel tool combining FM single setup. approaches tested HL-60 cells lysosomal-associated protein 2 (LAMP-2) muscle from facioscapulohumeral dystrophy mouse model. Yielding results greatly expediting workflow, are great value those working field indispensible future users microscopy.