Fluoro styrylbenzene fsb congo red derivative

Formalin-fixed samples were embedded in paraffin using standard procedures. Sections (8 μm) were cut, photobleached using a multispectral LED lamp, deparaffinized, and then processed for immunohistochemistry. Sections stained with antibodies were pretreated by incubation in heated 0.01M sodium citrate buffer as an epitope retrieval step. Following blocking with 10% normal goat serum, sections were incubated with primary antibody overnight at 4 °C (see Supp. Table 4 for antibody specfications). Primary antibody detection was performed using goat secondary antibodies with conjugated AlexaFluor 488 or AlexaFluor 647 (A-21245, Life Technologies). All immunohistochemistry-stained sections were subsequently stained with 2.5 μM fluoro-styrylbenzene (FSB; Congo red derivative) (Santa Cruz) in 1× PBS and propidium iodide (Sigma) washed before mounting. Samples were visualized using a 40× water-immersion lens (1.1 NA) or 63x oil-immersion (1.4 NA) in sequential scan mode on a Leica SP8 confocal microscope equipped with HyD detectors. 8-bit image z-stacks (1-μm steps) were collected at 1024 × 1024 or 2048 × 2048 pixel resolution. Images were processed using NIH ImageJ.