Wake Forest University School of Medicine

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets’ microenvironment in vitro and, potentially, in vivo settings.

The biology of intermuscular adipose tissue (IMAT) is largely unexplored due to the limited accessibility of human tissue. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing to identify the cellular composition of this unique adipose depot.