Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

Journal Article (Journal Article)

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.

Full Text

Duke Authors

Cited Authors

  • Kajimura, J; Ito, R; Manley, NR; Hale, LP

Published Date

  • February 2016

Published In

Volume / Issue

  • 64 / 2

Start / End Page

  • 112 - 124

PubMed ID

  • 26392518

Pubmed Central ID

  • PMC4812679

Electronic International Standard Serial Number (EISSN)

  • 1551-5044

Digital Object Identifier (DOI)

  • 10.1369/0022155415610792


  • eng

Conference Location

  • United States