Optimization of the tyramide signal amplification based fluorescence In situ hybridization in cleared thick brain tissues

Kumar V, Krolewski DM, Martin B, Parsegian A, Akil H, Watson SJ
Society for Neuroscience. 2016.


Tyramide signal amplification provides extraordinary detection sensitivity to fluorescence in situ hybridization (FISH) to visualize targets with low expression. It is based on catalyzed reporter deposition (CARD) technique where peroxidase mediated formation of highly reactive tyramide radicals result in the binding of fluorochrome-conjugated tyramide only at the site of the enzymatic reaction. This method has been successfully used for thin section FISH studies over the years, however it has been still a challenge to utilize this system with recently developed CLARITY and other tissue clearing methods for high resolution imaging and molecular characterization within intact large volumes. One of the major challenges of tyramide-based amplification in thick tissues (100 µm and more) is the rapid deposition of most of the available tyramide on the surface, exhausting the tyramide levels for deep layers of the tissue. Recent publications show a maximum signal penetration up to 100 µm depth using the commercially available tyramide kits. We explored this issue to achieve a uniform tyramide deposition across the tissue depth by using a combination of hydrolyzed cRNA probes and homemade biotin conjugated tyramide. Homemade biotinylated TSA is not only cost effective for larger tissue volumes but also provides an opportunity to regulate the catalysis/deposition of the tyramide. We characterized this improved method in fresh frozen rat- and CLARITY perfused mice-brain sections of various thickness. We are also comparing the mRNA detection in paraformaldehyde only fixed tissues (300 µm and 600 µm) and CLARITY tissues (1 mm) with or without 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC) fixation. We are further exploring the multi-target detection with and without TSA and were able to visualize good signal strength by using a simple immunohistochemical detection of biotin labeled probes for some of the abundantly expressed mRNAs.