Fontana-Masson Staining for the Detection of Melanin

The Fontana-Masson silver staining is described to be specific for melanin (Lillie, 1957). It is based on the argentaffin properties of melanin, i.e. the ability of melanin to reduce silver salts to form a metallic silver precipitate, which is easily detected in light and electron microscopy.

The stain produces the most reliable results on formalin-fixed paraffin sections (Figure 22.A).

Melanin appears blackish-brown and single particles can be distinguished from the SB up to the SC. Both kinds of frozen sections, without pre-treatment and IEM pre-fixed (B & C), also showed the characteristic staining but it was not possible to discern single particles as there were large accumulations of the black stain, especially in the SB and SSp (white arrows).

Especially the standard frozen sections displayed distinct staining in the upper epidermal layers, where little or no melanin would be expected in Caucasian skin (white arrows;

Figure 22.B). Post-staining with gold chloride has been omitted, as the staining results did not need blackening.

On resin sections, the Fontana-Masson staining produced a very detailed dark labelling for melanin that stood out well against the neutral red counterstain (Figure 22.D). This dark silver precipitate is quite precisely located in areas where melanin would be expected, i.e. mainly in the lower layers of the epidermis and forming caps above the nuclei (white arrowheads). But the dark labelling could not be observed when the counterstain was omitted, and on closer examination, a lot of the spots were stained dark red rather than black (black arrows), suggesting staining artefacts rather than specific labelling of melanin. The displayed sample was from an age spot (lesional), which explains the large amount of melanin in the SB.

The different staining results obtained for paraffin and frozen sections are remarkable, as the method is described to produce equally good results on frozen sections as on formalin-fixed paraffin sections, although the latter are to be preferred (Romeis, 1968; Churukian, 2002).

This might be explained by the differences in fixation, frozen sections being only postfixed in

Skin Pigment Characterization by Light and Electron Microscopy

Figure 22: Fontana-Masson staining for melanin. A black silver precipitate indicates the presence of melanin.

Neutral red was used as a counterstain. (A) Formalin-fixed, paraffin embedded section (4 µm) of Negroid skin.

(B) Frozen section (7 µm), postfixed with formalin before staining and (C) IEM pre-fixed frozen section (7 µm) of Caucasian skin. (D) High-pressure frozen, freeze-substituted sample of Caucasian skin, embedded in HM20 (500 nm section). The black silver precipitate is visible in all sections. The best results were produced on the paraffin sections. The frozen sections showed a slight overreaction (B and C; white arrows), while the resin sections produced only weak staining for melanin, that became visible only after counterstaining with neutral red. Some spots are stained dark red rather that black (D; black arrows). The white arrowheads indicate a few

intensely stained supranuclear melanin caps. Bars: 25 µm

PFA-fixative for 10 min, while the paraffin sections were treated with fixative for 24 hours.

The longer fixation time could be helpful. Aldehydes were found to enhance ammoniacal silver staining (Dion and Pomenti, 1983). Hence, the frozen sections would be expected to show less staining for melanin, rather than the achieved intensive stain. On the other hand, this might be an artefact introduced just by the fixation. To clarify this, frozen sections were processed omitting any chemical fixatives (data not shown), but the results were the same. It is also possible, that the omission of gold chloride post-staining yielded this over-staining.

Gold chloride is said to clear weakly impregnated substances (Burck, 1988).

The weak staining results obtained with resin sections can only be explained by masking-effects of the resin on sites of silver affinity, although the sections were etched with H2O2

Skin Pigment Characterization by Light and Electron Microscopy

prior to the incubation with the silver solution to remove the resin and uncover the affinity sites. But it is uncertain whether melanin retains its argentaffin properties after resin embed-ding. Nevertheless, longer incubation with the silver nitrate solution did not improve the staining results.

The over-staining of the frozen sections, as well as the doubtful staining results on resin sec-tions necessitated an assessment of the validity of Fontana-Masson’s silver staining regarding the quantitative analysis of the melanin content, or the amount of melanosomes within the epidermis respectively.

For this purpose, 10 µm frozen sections of Caucasian skin (Fitzpatrick’s skin phototype II) mounted on a glass slide, were treated according to the Fontana-Masson standard protocol (see chapter C.IV.2.3) followed by dehydration and room temperature embedding in Epon, positioning the skin section parallel to the surface of the resin bloc. After polymerization, the glass slide was removed. Consecutive sections were obtained for investigation in light and electron microscopy. 200 nm sections were mounted on a microscope slide and stained with toluidine blue to gain some contrast (see chapter C.IV.1.2). Subsequently 50 nm sections were obtained, collected on an EM-grid and stained with uranyl acetate for 45 min, followed by lead citrate for one minute. The results are displayed in Figure 23. The light microscopic images (A-C) confirm the presence of the silver precipitate at least in the lower epidermal layers (SSp-SB). Single black granules can be distinguished, mostly in cells of the SB. The characteristic supranuclear location of melanosomes was not apparent and sometimes the silver particles seem to be located underneath or even directly in the nucleus (white arrow-heads; Figure 23.B).

The TEM images were obtained in the area around the papilla left of enlargement B. The silver precipitate can be easily distinguished as round black granules, 8-30 nm in diameter.

Figure 23: Fontana-Masson benchmark. (A) 500 nm section of Caucasian skin frozen section, treated according to the Fontana-Masson staining protocol followed by embedding in Epon. Toluidine blue was used as counterstain. The black silver precipitate is visible in the basal cells (see enlargements B & C). The subsequent section was processed for TEM. The silver precipitate is observed throughout the epidermis. Dense accumulations are found on structures resembling melanosomes in shape and size (marked with black arrows).

But single silver particles are observed along the cell membranes and within the keratinocytes (black arrowheads). Loose accumulations of silver particles are frequently found with roundish shapes discernable beneath (white arrowheads). (D) Detail of SC. Single silver particles are abundant. (E) Detail of SG, and (F) detail of SSp. Loose silver accumulations are frequently found adjacent to cell borders. Residues of a desmosome are visible nearby (white arrow). (G) The largest amount of silver particles is located in the SB, partially in melanosome-resembling dense accumulations (black arrow), partially loosely scattered in the cells

Skin Pigment Characterization by Light and Electron Microscopy

Skin Pigment Characterization by Light and Electron Microscopy

The other structures appear more blurred in comparison due to poor contrast, as the tissue was not treated with heavy metals before embedding. Silver granules can be observed throughout the epidermis, with the largest amount of silver particles located in the SB. Dense aggrega-tions of precisely delineated shapes were found on dark, compact structures resembling melanosomes in shape and size (see black arrows, Figure 23.D, F & G). But less dense accumulations are frequently found in the SG and SSp, with roundish organelles discernable beneath, that are not markedly electron-dense, and that are most likely no melanosomes (white arrowheads, Figure 23.E & F). The shape and positioning adjacent to the cell mem-brane and the desmosome on the left, as well as residues of a memmem-brane opening into the intercellular space, suggest the roundish shape displayed in Figure 23.E to be a lamellar body rather than a melanosome. Additionally, single silver particles were abundantly distributed in the cytoplasm of keratinocytes and especially accumulated along cell membranes in the SC, even on the outside of the cells (black arrowheads, Figure 23.D & G). Figure 23.G shows a part of basal cell, with the nucleus (marked with N) visible on the left side. A large amount of silver aggregates are present, but again, the shapes are not always consistent with melano-somes. The accumulations are sometimes much larger than single melanosomes and a lot of single silver granules are visible within the cytoplasm and even in the nucleus.

These findings seem to confirm the assumption, that melanin is not the only substance that is stained by ammoniacal silver. Proteins in general bind low levels of ionic silver. Especially protein side chains consisting mainly of carboxylate-containing residues, thiols and thioether groups, can bind ionic silver. Aldehydes and ketones, including quinones, that are also present in melanin (see chapter B.I.2.1), react rapidly with ammoniacal silver (Lillie et al., 1957). The argentaffin reaction is also described for polyphenols, aminophenols and polyamins (Churukian, 2002). Melanin is most certainly not the only such substance in the human skin.

The longer the incubation with ammoniacal silver, the larger the chance of silver-binding to non-melanin compounds. On the other hand, Humbel et al. (1995) report that silver used as an enhancement agent for EM-immunolabelling experiments is not stable, but may be redistributed after exposure to the electron beam of the TEM. Thus, the distribution of single silver granules observed in the cytoplasm of corneocytes and cells of the SG may not repre-sent silver affinity sites, but simply relocated silver particles. However, the reaction is sus-ceptible to artefacts, any contamination of the utilized glassware or reagents must be carefully avoided, as they also give rise to silver precipitates (Burck, 1988). Any variations in the reac-tion condireac-tions, e.g. pH, composireac-tion and temperature of the reacreac-tion medium, considerably

Skin Pigment Characterization by Light and Electron Microscopy affects the quantitative characteristics of this staining method (Gallyas, 1979).

The false-positive staining observed in the TEM images may be considered negligible and too small to be detected in LM. But as the standard application of this staining method is on LM sections, the thickness of the investigated section must be considered, as well as the strong light scattering property of silver colloids. The TEM sections are 200 nm thick, while sections of LM are usually 4-7 µm thick. The thicker the section, the more blurred the resulting image, because of overlapping structures, and thus the false-positive staining is summed up and becomes visible also in LM images. This may lead to overvaluation of melanin content of a sample.

If the method is to be used for quantitative analysis, it is therefore crucial to standardize incu-bating solutions and incubation time, in order to – at least – minimize inter-experimental variations. Extensive controls are necessary to assess the degree of false-positive staining.

Qualitative comparison of the melanin content of different skin samples is certainly possible.

But with regard to the possible overassessment, quantitative analysis seems not to be appropriate.