Abstract
Micro-computed tomography (micro-CT) scanners allow for the three-dimensional analysis of bone structure to be visualized and numerous bone structural parameters to be quantified with a high degree of accuracy. Nucleic acid-based immunohistochemistry (IHC) allows for characterization of the presence, amounts and different types of DNA in paraffin-embedded fixed tissue sections. Micro-CT scanners (i.e., Bruker SkyScan 1173 µCT system), along with immunohistochemically processed tissue sections were used to examine a variety of different fixatives in order to characterize the effects of fixation on bone morphology (i.e., adult chicken), and preservation of different types of intact, non-denatured nucleic acids (e.g., B-DNA, Z-DNA). Several different types of bone (e.g., femur, tibia, and vertebra) were characterized. Fixation was performed using 10% Neutral Buffered Formalin (NBF), and Formalin-Alcohol-Acetic Acid (FAA) for 24, 48, 72 and 96 hours at room temperature. Both decalcified and non-decalcified bone tissues were processed. Molecular biological grade fixatives and water were used in order to ensure the best histotechnological results. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact B-DNA and Z-DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA. Tissues processed in 10% NBF required antigen retrieval. Our group is correlating morphological differences observed with different fixatives using a microCT scanner, relative to DNA identification employing immunohistochemical staining of bone tissue sections with anti-B-DNA and anti-Z-DNA monoclonal antibodies. Our data demonstrates that FAA results in easier histotechnological processing of bone tissue, better morphological results and superior IHC staining. Research project was supported by an NSF grant, Award ID No: 1828305. .
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