Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique

Abstract

The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Structural and antigenic preservation of plant samples by microwave‐enhanced fixation, using dedicated hardware, minimizing heat‐related effects

“…While the reason why microwave irradiation of a polar substance results in energy absorption and heating is evident and the physical mechanisms that are responsible for heat production are well understood (Boon and Kok, 1988), it is not clear whether heating is the only cause of the enhancing effect of microwaves on fixation or if some additional effects are involved and, in this case, what might be their nature. In fact, this remains the main open discussion on this topic (Feirabend et al, 1992;Kok and Boon, 1990b;Leonard and Shepardson, 1994;Medina et al, 1995;Wild, 1991). Otherwise, an excess of heat applied to the sample by microwave irradiation is commonly considered unacceptable, since it results in protein denaturation; in general, published procedures of microwave-enhanced fixation recommend keeping the temperature below 50°C (see also Moriguchi et al, 2002).…”

A rapid alternative technique for obtaining silver-positive patterns in chromosomes

“…In the diagnosis of pathogenic tumors, the utilization of microwave irradiation shortened the time needed to evidence the nucleoli (Medina et al, 1995;Li et al, 1995) by up to one minute or close to it. Similarly, the exposure of the slides to microwaves drastically reduced the time needed for Ag-NOR detection, as they could be evidenced in approximately 5 s.…”

“…In the latter, the mean number of Ag-NORs is used as a pathological marker for some malignant tumors. As an alternative to the standard procedures, it was suggested to utilize microwave irradiation for nucleolus localization in histological preparations, in order to fasten and improve the diagnosis (Medina et al, 1995;Li et al, 1995).…”

A new version of the Ag−NOR technique. A combination with DAPI staining

“…In interphase cells, fibrillar constituents of the nucleoli (their fibrillar centres and dense fibrillar components), or whole nucleoli are distinctly Ag-NOR-positive (Ploton et al, 1987;Moreno et al, 1988;Roussel et aI., 1992;Medina et al, 1995). Cytological and immunolocalization studies have shown that the Ag-NOR-positive material consists of acidic proteins associated with ribosomal DNA (rDNA) (e.g., Goodpasture & Bloom, 1975;Lischwe et al, 1979;Howell, 1982).…”

“…Several modifica-BILINSKI and BILII~ISKA tions of the technique for various biological applications have recently been suggested (e.g. Lindner, 1993;Medina et al, 1995;Li et al, 1995).…”