A Comparison of Two Methods for Colorimetric in situ Hybridization Using Paraffin-Embedded Tissue Sections and Digoxigenin-Labeled Hybridization Probes

Abstract

Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90–100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals.

Received September 27, 2012; accepted February 22, 2013

ACKNOWLEDGMENTS

I thank the histology staff at the Oxford Cooperative Laboratory for their assistance in preparing the tissue sections. Gretchen Messick and Kathy Tang provided the tissue sections of crab tissues infected with the blue crab reovirus and the reovirus probe. Vicki Blazer and Delores Baxa provided the tissue sections of fish and Tubifex worms infected with Myxobolus cerebralis.