ABSTRACT
The choice of fixation method significantly impacts tissue morphology and protein visualization after immunohistochemistry (IHC). In this study, we compared the effects of paraformaldehyde (PFA) and trichloroacetic acid (TCA) fixation prior to IHC on chicken embryos. Our findings underscore the importance of validating fixation methods for accurate interpretation of IHC results, with implications for antibody validation and tissue-specific protein localization studies. We found that TCA fixation resulted in larger and more circular nuclei compared to PFA fixation. Additionally, TCA fixation altered the appearance of subcellular localization and fluorescence intensity of various proteins, including transcription factors and cytoskeletal proteins. Notably, TCA fixation revealed protein localization domains that may be inaccessible with PFA fixation. These results highlight the need for optimization of fixation protocols depending on the target epitope and model system, emphasizing the importance of methodological considerations in biological analyses.
ABSTRACT
Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.
