Identification of Melanocytes by Immunohistochemistry

Immunohistochemistry presents another possibility to detect melanosomal proteins, which has several advantages compared to histochemical reactions. Immunolabelling is based on highly specific binding of IgG-antibodies to target proteins. Direct or indirect labelling of these anti-bodies with fluorescent dyes enables very precise detection of the target proteins by fluores-cent light microscopy and CLSM. In addition, by double labelling with a combination of

Skin Pigment Characterization by Light and Electron Microscopy complementary antibodies an explicit identification of melanocytes and further information, e.g. on morphology and location can be achieved.

Several antibodies were tested, directed either against melanosomal enzymes or melanocyte-specific components. The staining results of most of the antibodies are dependent on the pre-treatment of the sample. Any kind of fixation – chemical or physical – influences structure and properties of the target proteins and therefore their availability for antibody-binding. This conformational change may be necessary to make the antigen available for the antibody, but it may also result in total masking or destruction of the antigen (Griffiths, 1993; Hayat, 2002).

Some antibodies, e.g. both tested S100 antibodies or the actin-labelling toxin phalloidin, worked only on chemically pre-fixed frozen sections, while others (e.g. α-EEA1 or the Mela-noma antibodies) produced results only when fixatives were avoided. Only a few antibodies, i.e. α-collagen IV, α-keratin and Mel-5 are independent of sample pre-treatment. Some anti-bodies, like α-clathrin, α-tyrosinase or α-tubulin showed no staining at all. A full list of tested antibodies and the achieved staining results in dependence of the type of frozen section is displayed in Table 12. The optimal working dilution was determined individually for each antibody based on recommendations of the supplier. The applied dilutions are listed in chapter C.IV.3.

Table 12: Staining results of all tested antibodies depending on the pre-treatment of the sample.

all kinds of frozen sections

frozen sections, no pre-treatment

frozen sections,

chemical fixation no staining results

α-Collagen IV α-Laminin 2 α-EEA1 α-Clathrin

α-Keratin α-Vimentin α-S100 α-MiTF

Mel-5 Melanoma Ab-2 Phalloidin α-Tubulin

Melanoma Ab-3 α-Tyrosinase

Melanoma Ab-5 Mel-1

Melanoma Ab-1

For the identification of melanocytes, a combination of two antibodies was employed: an antibody labelling one enzyme involved in melanosome formation or melanin polymerisation was combined with markers for specific components of the cytoskeleton or melanocyte-associated proteins that are not directly involved in melanogenesis. But due to the differences in the affinity for the type of frozen sections, the range of combinations is restricted. Suitable antibody combinations are listed in Table 13.

Melanoma Ab-2 (HMB50) and Ab-5 (NKI/ beteb) are two different clones of antibodies

Skin Pigment Characterization by Light and Electron Microscopy

directed against gp100, an enzyme that promotes the biogenesis of premelanosomes. The staining results of both antibodies are comparable. Melanoma Ab-3 is a mixture of Ab-1 (clone HMB45) and Ab-2 and is not described, as the results are identical to Ab-2. HMB45 is described to label epidermal and dermal melanoma cells, but no melanocytes in normal adult epidermis (Smoller et al., 1991). Accordingly, Ab-1 did not show any labelling results.

Table 13: Antibody combinations for the description of melanocytes. Only the antibodies, which showed reproducible, specific labelling results are listed.

Mel-5

(α-TRP1) Melanoma Ab-2

(α-gp100) Melanoma Ab-5

(α-gp100)

α-Collagen IV ++ ⁽³⁾ + ⁽²⁾ ++ ⁽³⁾

α-EEA 1 + ⁽¹⁾ - -

α-Keratin ++ ⁽³⁾ + ⁽²⁾ + ⁽³⁾

α-Laminin 2 + ⁽²⁾ + ⁽²⁾ + ⁽²⁾

α-S100 ++ ⁽¹⁾ - -

α-Vimentin + ⁽²⁾ + ⁽²⁾ +

Phalloidin ++ ⁽¹⁾ - -

(1) these combinations work only for chemically pre-fixed frozen sections

(2) these combinations require frozen sections without chemical fixation

(3) these combinations work well for both, frozen sections with, and without chemical pre-fixation (++) intensive labelling (+) moderate labelling (-) combination not possible

Mel-5 labels another enzyme involved in melanin polymerisation, i.e. TRP-1, which is active in melanosome stages III and IV (Bhawan, 1997). This antibody was found to be the most sensitive of the tested melanocyte markers. A direct comparison of the three antibodies ap-plied to serial sections is displayed in Figure 16. All three antibodies detect melanocytes in corresponding locations of the sample, but the amount of details varies, Melanoma Ab-2 (Figure 16.A) displaying the least and Mel-5 the most details (Figure 16.C). This findings are in accordance with Dean et al. (2002) who also found Mel-5 to be the most sensitive of melanocyte markers. In addition, this antibody is not dependent on sample pre-treatment and produced very good staining results at high dilutions. Therefore, Mel-5 was used as melano-cyte marker for further investigations.

Nevertheless, Mel-5 recognises a protein that is found active in melanosome stages III and IV.

As melanosomes at this stage of development are already transferred to keratinocytes, it was necessary to determine, whether this antibody detects melanocytes only, or whether any melanosomes within keratinocytes, that still exhibit TRP-1 activity are recognized as well.

Skin Pigment Characterization by Light and Electron Microscopy

Figure 16: Comparison of antibodies directed against melanosomal proteins. Consecutive serial sections of epidermis including a hair follicle (asterisk). Melanocytes are displayed in green, nuclei in blue. (A) Staining result for Melanoma Ab-2, an antibody directed against gp100, an enzyme involved in premelanosome formation (clone HMB50). Melanocytes can be distinguished in the basal layer of the epidermis, as well as in the hair follicle. (B) Staining result for Melanoma Ab-5 (clone NKI/beteb) directed against gp100. Melanocytes are detected in places corresponding to (A), but more details are visible. (C) Melanocytes are labelled with Mel-5.

This antibody recognizes TRP1, an enzyme involved in melanin polymerization, found in late developmental stages of melanosomes. Again, melanocytes are detected in places corresponding to the previous section. This

antibody provides the most detailed staining. Bars: 25 µm

Hence, additional properties of melanocytes, such as the cytoskeleton, were included in the investigation. The intermediate filaments of melanocytes are made of vimentin, while the keratinocytes contain keratin. An α-vimentin antibody can therefore be used as a direct marker, while α-keratin provides an indirect marker, i.e. a negative image of melanocytes.

Another direct marker is the early endosome marker α-EEA1, as melanosomes derive from early endosomes (Wilson et al., 2000; Raposo and Marks, 2002). The S100 protein is present in all cells derived from the neural crest and detects any kind of cells forming dendrites (Donato, 2003). In combination with a melanogenesis-associated marker, α-S100 provides clear identification of melanocytes as well as a means to describe their morphology.

Antibodies directed against components of the basement membrane, like collagen IV and α-laminin 2, as well as the cytoskeleton marker phalloidin can be employed to describe the positioning of melanocytes in the epidermis and their interactions with keratinocytes.

Figure 17 exhibits examples for double labelling with Mel-5 to identify melanocytes (Mel-5 labelling is displayed in green). Mel-5 and α-keratin show complementary staining patterns, α-keratin provides a negative image of the melanocytes and no colocalisation is observed (Figure 17.A). The intermediate filaments of melanocytes were labelled with α-vimentin (Figure 17.C). The antibody displayed only a low affinity for epidermal cells, while dermal cells like fibroblasts were clearly stained. Yet Mel-5 positive cells also show small spots of colocalisation with α-vimentin (marked with white arrowheads). Double labellingwithα-S100

Skin Pigment Characterization by Light and Electron Microscopy

Figure 17: Antibody combinations for the identification and description of melanocytes. Frozen sections without chemical pre-fixation were used for A & C, and with IEM pre-fixation for B & D. The melanocyte marker Mel-5 is displayed in green, colocalisation with the respective marker appears yellow. The SC is positioned at the top of the images, the dotted white line represents the basement membrane. (A) Double labelling for Mel-5 and α-keratin. All Mel-5 positive cells are negative for α-keratin and v.v. (B) Mel-5 combined with a marker for dendritic cells, α-S100. Cells that show double labelling (arrowheads) are clearly identified as melanocytes. (C) Double labelling for Mel-5 and α-vimentin. The cytoskeleton of melanocytes contains vimentin instead of keratin, while keratinocytes do not contain vimentin. The α-vimentin antibody labels mostly dermal cells, i.e. fibroblasts, but a few epidermal cells are positive for vimentin (arrowheads). (D) Combination of Mel-5 and α-EEA1 (early endosome marker) labelling. Premelanosomes are of endosomal origin, and α-EEA1 is present in melanocytes. Cells positive for both markers are indicated by white arrowheads.

α-EEA1 displays unspecific labelling in keratinocytes surrounding a hair follicle (asterisk). Bars: 25 µm

revealed several dendritic cells in the upper layers of the epidermis (Figure 17.B). In the SB, most of the stained cells are positive for both, Mel-5 and α-S100 (marked with white

arrow-Skin Pigment Characterization by Light and Electron Microscopy This staining result underlines the positioning of melanocytes along the basement membrane.

The double staining with Mel-5 and α-EEA1 shows colocalisation in most of the recognized cells (Figure 17.D, indicated by white arrowheads). The intracellular location of the α-vimentin and α-EEA1 labelling is near the nuclei, as would be expected. Hence, these antibodies can be used for the identification of melanocytes only, but not for the description of their morphology and interactions with keratinocytes. In contrast, Mel-5 and α-S100 stain cytoplasm and dendrites of the melanocytes.

These findings confirm the specificity as well as the high sensitivity of Mel-5 as a suitable marker for melanocytes. In combination with α-collagen IV, Mel-5 provides detailed infor-mation on morphology and interaction of melanocytes with keratinocytes (Figure 18). This combination was used to determine the density and distribution of melanocytes and describe the epidermal melanin unit (see chapter II.1).

Figure 18: Double labelling for Mel-5 and α-collagen IV. Melanocytes are represented in green, the basement membrane (labelled with α-collagen IV) in red and nuclei (labelled with Sytox Green) are displayed in blue. This antibody combination is useful to describe the exact localisation of melanocytes relative to the basement membrane and their density in the epidermis (see chapter E.II: Melanocyte Distribution ). Bar: 50 µm