MicroCT Scanner to Differentiate Histotechnological Processing of Bone Tissue Using Different Nucleic Acid‐Based Fixatives

Abstract

Micro‐computed tomography (μ‐CT) scanners permit researchers to characterize the three‐dimensional structure of bone. They allow for the visualization of many bone structural parameters to be quantified with an extremely high degree of precision. Immunohistochemistry (IHC) (colorimetric) and immunofluorescence (IF) allows for characterization of DNA and RNA in fixed tissue sections. μ‐CT scanners along with IHC and IF allows processed tissue sections to be examined by a variety of different fixatives, in order to characterize the effects of fixation on bone morphology (i.e., adult chicken and mice), and preservation of different types of intact, non‐denatured nucleic acids (e.g., right‐handed B‐DNA, left‐handed Z‐DNA). Fixation was performed using 10% Neutral Buffered Formalin (NBF), Formalin‐Alcohol‐Acetic Acid (FAA), Davidson’s fixative, and Carnoy’s solution for 72 hours at room temperature. Both decalcified and non‐decalcified bone tissues were processed. Ultra‐pure molecular biological grade fixatives and water were used in order to ensure the best histotechnological data. Tissues processed in FAA resulted in hardening of tissue, but good fixation of intact DNA. Tissues processed in 10% NBF resulted in softer tissue, but poor fixation of intact DNA and required antigen retrieval (AR). Tissues processed in Carnoy’s solution resulted in hardened tissue, but excellent DNA IHC and IF. Tissues processed in Davidson’s fixative resulted in hardened tissues, but good preservation of DNA. We will try to correlate structural bone differences observed with the μCT scanner, relative to DNA isolation and IHC and IF staining of bone tissue sections with anti‐B‐DNA and anti‐Z‐DNA monoclonal antibodies. These results will better enable researchers to histotechnologically process tissue while obtaining data on DNA structure and function.