Using Enzyme-Linked Markers with Chromogenic Substrates in Expansion Microscopy

Expansion microscopy (ExM) is a methodology which increases the imaging resolution of sample through expansion prior to optical by crosslinking it hydrogel, digesting it, and swelling gel, thereby expanding sample. Though many variations ExM have been created, all these rely on labeling with fluorescent markers. Here we use enzyme-linked antibody markers generate chromogenic staining proteins in myofibrils. Immunohistochemistry microscopic samples widely used for medical diagnostic procedures as well research can significantly broaden applications this technique. As an example method, myosin labeled primary antibody, embedded polyacrylamide digested using Proteinase K. After digestion, gel incubated secondary conjugated either alkaline phosphatase or peroxidase, then expanded washing water. Chromogenic substrates are applied triggered near myosin, observed at A-bands myofibrils brightfield microscopy. This allows be towards independent from fluorescence Super-resolution observation internal structures within sarcomere, especially thick filaments, transmitted light facilitates identification structural heterogeneities that reveal molecular interactions other myosins binding proteins. method improves both lateral axial therefore unique form super-resolution (Supported NIH/NHLBI R01HL149164.)