title: Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.
abstract: 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.
authors.affiliation: Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan (JK, RI)
authors.forename: Junko
authors.initials: J
authors.lastname: Kajimura
authors.affiliation: Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hiroshima, Japan (JK, RI)
authors.forename: Reiko
authors.initials: R
authors.lastname: Ito
authors.affiliation: Department of Genetics, Paul D. Coverdell Center, 500 DW Brooks Drive, University of Georgia, Athens, Georgia (NRM)
authors.forename: Nancy R
authors.initials: NR
authors.lastname: Manley
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina (LPH)
authors.forename: Laura P
authors.initials: LP
authors.lastname: Hale
mesh_terms: D000293:Adolescent; D000328:Adult; D001391:Azo Compounds / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008875:Middle Aged; D009281:Naphthalenes / Q000737:chemistry; D061848:Optical Imaging; D010232:Paraffin / Q000737:chemistry; D016612:Paraffin Embedding / Q000379:methods; D013950:Thymus Gland / Q000648:ultrastructure*; D016707:Tissue Fixation / Q000379:methods; D055815:Young Adult
chemical_list: D001391:Azo Compounds; D004396:Coloring Agents; D009281:Naphthalenes; D005557:Formaldehyde; D010232:Paraffin; C016118:Sudan Black B
keywords: archival tissue; image analysis; immunofluorescence; thymus; tissue processing