Combined In Situ Hybridization and Immunohistochemistry in Rat Brain Tissue Using Digoxigenin-Labeled Riboprobes

This protocol describes a procedure for combining non-radioactive in situ hybridization (ISH) histochemistry with multi-label fluorescence immunohistochemistry (IHC) on rat brain tissue sections. This allows visualization of multiple mRNA and protein targets located within the same or different subcellular compartments. A comprehensive description of RNA probe preparation, validation and storage conditions is described. This is followed by the tissue preparation and tissue processing procedure for combined ISH/IHC on free-floating brain sections. Both the tissue and the RNA probes must be prepared and handled under strict RNase-free conditions up until the point of RNA hybridization.