Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues

Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more proteins within tissue sections. The protocol can be applied to other tissue types. For complete details on the use and execution of this protocol, please refer to Gonzalez Abreu et al. (2022). [Display omitted] •Multiplex fluorescent IHC allows for simultaneous visualization of multiple proteins•A protocol for immunolabeling proteins in free-floating rodent brain tissue•The free-floating approach results in less tissue loss and greater antibody penetration•Can be applied to brain tissue of different vertebrates or other tissue types Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more proteins within tissue sections. The protocol can be applied to other tissue types.