RERF Report No. 10-15

Paper published in Journal of Histochemistry & Cytochemistry §“Optimization of Dual-Color Immunofluorescence Protocols for Formalin-Fixed,Paraffin-Embedded Archival Tissues”
Junko Kajimura, Reiko Ito, Nancy R. Manley, Laura P. HaleJ Histochem Cytochem 2016 (February) 64: 112-24(doi: 10.1369/0022155415610792)

Study Findings
We developed a protocol for producing high-contrast, high-quality immunofluorescence
images from formalin-fixed paraffin-embedded (FFPE) thymus blocks prepared up to 60 years
ago. This dual-color immunofluorescence protocol enables state-of-the-art archival-tissue imageanalyses. The protocol also enables pathological analyses using smaller sample quantities. Itwill thus be instrumental in studies that use precious specimens obtained from A-bomb
survivors.
Explanation
Archival FFPE tissues are generally unsuited for immunofluorescence staining because oftissue quality degradation and background noise from autofluorescence. This was particularly
true for FFPE tissues from A-bomb survivors stored at RERF for as long as 60 years. Dr. Junko
Kajimura, research scientist, RERF Department of Molecular Biosciences, et al. recently
developed a combination of tissue processing and staining techniques that minimize noise fromautofluorescence* and maximize fluorescence signals from cells under study. We also developed
a dual-color immunofluorescence protocol that, with the latest digital image processing
techniques, provides images of high contrast and quality.
* Autofluorescence: Intrinsic fluorescence from cells and tissues and sometimes difficult todistinguish from fluorescence of stained cells under study.

1. Objectives
The objective was to develop a dual-color immunofluorescence protocol suitable foranalyzing the FFPE tissue specimens of A-bomb survivors stored at RERF.
2. Methods
Using 10 FFPE archival thymus tissue specimens from A-bomb survivors in Hiroshima
prepared between 1954 and 1970, we employed the dual-color immunofluorescence protocol tostain thymic epithelial cells with anti-CK14 antibody* and cortical thymocytes with anti-CD1aantibody.* Attempting to improve the staining and image analysis techniques, we (1) included
multiple independent steps to enhance the specificity of immunostaining; (2) used chemical
processing procedures to reduce noise from autofluorescence without losing fluorescencesignals in tissues; and (3) produced high-quality, high-contrast fluorescent images provided by
the fluorescence microscope’s digital image processing functionality.
*Anti-CK14 antibody: An antibody that reacts with type I keratin expressed in the cytoplasm ofthymic epithelial cells (CK14)*Anti-CD1a antibody: An antibody that reacts with a membrane glycoprotein expressed on
cortical thymocytes (CD1a)3. Results
We obtained high-quality, high-contrast, low-noise dual-color fluorescent images of1

RERF Report No. 10-15

thymic epithelial cells and cortical thymocytes from old archival FFPE thymus tissues through(1) the inclusion of multiple independent steps for tissue processing and staining; (2) treatment
with NH3/EtOH* and removal of autofluorescence with Sudan Black B*; and (3) enhancement ofthe signal-to-noise ratio using digital image processing.
*Treatment with NH3/EtOH: Tissue sections are treated with a mixture of ammonia (NH3) andethanol (EtOH) to remove cross-links (stable bonds of proteins) formed by formalin fixation,
expose amino acids comprising antigenic determinants, and consequently increase theirreactivity with antibodies.
*Removal of autofluorescence with Sudan Black B: Staining FFPE tissue sections with 0.3%
Sudan Black B reduces autofluorescence (noise) attributable to blood cells or elastin fibers in
FFPE tissue sections without affecting fluorescence signals from fluorescent-labeled antibodies.

Significance of the Study
The improved protocol we developed provided high-quality dual-color fluorescent images
using FFPE tissue specimens (thymic epithelial cells and cortical thymocytes) stored up to 60years in the past. This protocol is considered to be suitable also for immunofluorescencestaining of other types of archival specimens besides thymus tissues.

The Radiation Effects Research Foundation has studied A-bomb survivors and their offspring inHiroshima and Nagasaki for more than 60 years. RERF’s research achievements are considered theprincipal scientific basis for radiation risk assessment by the United Nations Scientific Committee on theEffects of Atomic Radiation (UNSCEAR) and for recommendations regarding radiation protection
standards by the International Commission on Radiological Protection (ICRP). RERF expresses its
profound gratitude to the A-bomb survivors and survivors’ offspring for their cooperation in our studies.
§Journal of Histochemistry & Cytochemistry is a peer-reviewed scientific journal of cell biology
established in 1953. It covers research into the structure and function of cells, tissues, and organs as wellas components of development, differentiation, and disease, in addition to microscopy and imaging
techniques. (Impact factor for 2014: 1.959)

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