Optimized protocol for retinal wholemount preparation for imaging and immunohistochemistry Academic Article uri icon

Overview

MeSH Major

  • Immunohistochemistry
  • Retina
  • Tissue Fixation

abstract

  • Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing. Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

publication date

  • December 13, 2013

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC4048354

Digital Object Identifier (DOI)

  • 10.3791/51018

PubMed ID

  • 24379013

Additional Document Info

start page

  • e51018

number

  • 82