Detection of Proliferating Cell Nuclear Antigen in Tissues of Three Small Fish Species

Abstract

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing, and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin, but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis, ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.

Dietary influences over proliferating cell nuclear antigen expression in the locust midgut

“…Clone PC10 has been successfully used in many studies of cell proliferation in mammals (Hall et al, 1990;Preziosi et al, 1995;Start et al, 1992). It has also been reported that clone PC10 gives the best results in PCNA detection in fish tissues (Ortego et al, 1994). The only study on immunological PCNA detection in insect tissue sections to our knowledge also used clone PC10 as the primary antibody (Yamaguchi et al, 1991).…”

Stage and season effects on cell cycle and apoptotic activities of germ cells and Sertoli cells during spermatogenesis in the spiny dogfish (Squalus acanthias)

“…Immunostaining was carried out using the Unitect Avidin-Biotin-Peroxidase Immunohistochemistry Detection System Kit (Oncogene Science, Cambridge, MA, USA), which contains the recombinant PCNA mouse monoclonal PC10 antibody shown to interact with fish PCNA antigen (Ortego et al 1994). The procedure was a modification of that suggested by the supplier.…”

“…Endogenous peroxidase was quenched by treating sections in darkness for 10 min with 3% (v/v) hydrogen peroxide. Following a rinse in distilled water, the sections were subjected to an antigen-retrieval procedure (Ortego et al 1994), which involved heating in a microwave oven (450 W) in 1% (v/v) anhydrous zinc sulphate for 3 min, resting for 1 min and heating a further 3 min. After cooling for 15 min, sections were rinsed in distilled water and placed in 0.5% (v/v) Tween 20 in PBS (137 mM NaCl, 29 mM NaH 2 PO 4 ·H 2 O and 9 mM Na 2 HPO 4 , pH 7.4; referred to here as PBS-A) for 5 min.…”

Stage-dependency of apoptosis and the blood-testis barrier in the dogfish shark (Squalus acanthias): cadmium-induced changes as assessed by vital fluorescence techniques

“…containing the recombinant PCNA mouse monoclonal PC10 antibody shown to interact with fish PCNA antigen (Ortego et al 1994). In brief, endogenous peroxidase was quenched by treating sections in darkness for 10 min with 3% hydrogen peroxide.…”

“…In brief, endogenous peroxidase was quenched by treating sections in darkness for 10 min with 3% hydrogen peroxide. Following a rinse in distilled water, the sections were subjected to an antigen retrieval procedure (Ortego et al 1994) that involved heating the sections in a microwave oven (450 W) in 1% anhydrous zinc sulphate for 3 min, resting for 1 min, and heating for a further 3 min. After being cooled for 15 min, sections were rinsed in distilled water and placed in 0.5% Tween 20 in phosphate-buffered saline (PBS-A: 137 mM NaCl, 29 mM NaH 2 PO 4 •H 2 O, 9 mM Na 2 HPO 4 , pH 7.4) for 5 min.…”