SEARCH

SEARCH BY CITATION

Keywords:

  • immunohistochemistry;
  • melanocytes;
  • tissue in situ hybridization

Abstract:

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players as they produce and distribute melanin that protects the skin from ultraviolet (UV) radiation. In vitro experiments on melanocytic cell lines are useful to study melanogenesis and their progression towards melanoma. However, interactions of melanocytes with keratinocytes and with other types of cells in the skin, such as fibroblasts and Langerhans cells, are also crucial. We describe two techniques, immunohistochemistry (IHC) and tissue in situ hybridization (TISH), that can be used to identify and study melanocytes in the skin and their responses to UV or other stimuli in situ. We describe a practical method to localize melanocytic antigens on formalin-fixed, paraffin-embedded tissue sections and in frozen sections using indirect immunofluorescence with conjugated secondary antibodies. In addition, we detail the use of TISH and its combination with IHC to study mRNA levels of genes expressed in the skin at cellular resolution. This methodology, along with relevant tips and troubleshooting items, are important tools to identify and study melanocytes in the skin.


Introduction

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

Skin is a complex structure providing important critical functions. Not only does it serve as an essential barrier from environmental stress, but also as a key organ for contact and exchange between the organism and its environment. Cells in the skin are certainly the most exposed to external stimuli. Therefore, they have developed complex communications and tight regulations to respond to such stimulations. Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players as they produce and distribute melanin, which protects the skin from ultraviolet (UV) radiation. Melanocytes are also the type of cell from which melanoma, one of the deadliest cancers, is derived. In vitro experiments on melanocytic cell lines are helpful to study melanogenesis and the progression of melanocytes towards melanoma. However, interactions of melanocytes not only with keratinocytes but also with other types of cells in the skin, such as fibroblasts and Langerhans cells, also appear to be crucial (1–4). Fortunately, thanks to its superficial localization, the skin is relatively easy to biopsy and thus allows for in vivo studies. However, since the first description of pigment cells in 1819 by Sangiovanni, the study of melanocytes within the skin was barely impossible (see Ref. 5 for review). In 1917, Bloch introduced the 3,4-dihydroxyphenylalanine (DOPA) reaction to analyse melanin-producing cells. More than 30 years after, Fitzpatrick et al. showed the activity of tyrosinase (TYR) in normal human skin after UV irradiation in vivo using the DOPA staining (6). Nowadays, the wide variety of specific antibodies allows studying in vivo protein expressions using immunohistochemistry (IHC). More recently, tissue in situ hybridization (TISH) completes our technical tools to analyse RNA expression within the skin.

The aim of this review was to be very practical and to outline approaches that can be used to specifically identify melanocyte subpopulations in the skin. We focus on two techniques that are commonly used in our laboratory to identify and study melanocytes in the skin: IHC and TISH.

Interfollicular and follicular melanocyte markers

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

Melanocytes are present not only in the epidermis and in hair follicles but also in the eye, inner ear and leptomeninges. They can also be found in the Harderian gland and even in some mesenchymal tissues. However, we will limit this review to melanocytes localized in the skin.

By virtue of their specific function (melanogenesis), melanocytes can be identified by a growing number of markers specifically expressed by those cells, which are usually involved with that specific function. As examples, TYR and microphthalmia-associated transcription factor (MITF) are two of the most commonly used specific biomarkers for evaluating the regulation of the melanogenic system after environmental stimulation. TYR, a membrane glycoprotein, was the first melanogenic enzyme identified as important for melanin synthesis (7–9). In the melanin biosynthetic pathway, TYR catalyses the rate-limiting step of hydroxylating the amino acid tyrosine to l-DOPA. Incubations of fixed paraffin sections with DOPA can lead to melanin production which is readily visible in tissue sections and is quite useful to identify melanocytes (10). MITF is considered the principal transcription factor in charge of regulating melanocyte function (11–13). It moderates several melanocyte-specific genes that encode melanosomal proteins such as TYR, dopachrome tautomerase (DCT) and MART1 (melanoma antigen recognized by T cells). DCT is the enzyme that catalyses the tautomerization of the melanogenic intermediate DOPAchrome to 5,6-dihydroxyindole-2-carboxylic acid (14). Importantly, DCT is one of the earliest melanocyte markers expressed and is detected both in melanocytes and in melanoblasts (15). MART1 is yet another melanosomal protein that is also a melanoma-specific target for tumor-directed T lymphocytes and it continues to be studied as a potential target for immunotherapy of melanoma (16). Thus, the expression of MART1 is useful as a specific marker to localize melanocytes. MART1 forms a complex with gp100 (Pmel17/silver) and influences its expression, therefore modifying the process necessary for melanosome maturation and structure (17). Pmel17/gp100 is an essential protein required for formation of the structural matrix of stage II melanosomes (18).

Melanocytes located in the basal layer of the epidermis are quite similar to those located in the basal layer of the hair follicle infundibulum (Fig. 1a). They are also similar to melanocytes located amongst the basal sebocytes of the sebaceous gland, even if those are only weakly to moderately pigmented. Another subpopulation of melanocytes is located in the mid-portion of the hair follicle outer root sheath (the so-called ‘bulge’ region). Melanocytes in this region are poorly differentiated, non-pigmented, and are usually considered melanocyte stem cells. The most proximal follicular melanocyte subpopulation, which contributes to pigmentation of the hair shaft, is located in the hair bulb above and around the mid-upper follicular papilla. Anagen hair bulbs also contain poorly differentiated melanocytes. Those amelanotic hair bulb melanocytes may represent ‘transient’ melanocytes that are migrating from precursor melanocytes stored in the upper outer root sheath (19). The antigenic expression patterns of markers for these various melanocyte subpopulations have been widely explored (20–24), and antibodies or specific RNA probes can be used to define the subpopulations of melanocytes in the epidermis and in hair follicles (summarized in Fig. 1c). The subcellular localization of melanocyte-specific antigens is summarized in Fig. 1b.

image

Figure 1.  Schematic of melanocyte populations within the skin and specific antigens. (a) Localization of melanocyte populations in the skin, and (b) subcellular localizations of melanocyte-specific antigens. (c) Table summarizing antigens and their expression patterns that allow identification of melanocyte subpopulations.

Download figure to PowerPoint

Immunohistochemistry

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

Localizing specific melanocyte subpopulations in murine and human tissues can be accomplished using standard IHC techniques. A practical method to localize melanocytic antigens in formalin-fixed, paraffin-embedded tissue sections and in frozen sections using indirect immunofluorescence with conjugated secondary antibodies is presented schematically in Fig. 2 and is detailed in Table 1. Endothelin 1 has been implicated in the response of UV and appears also to play a role in pigmentation in basal cell carcinoma (25). Figure 3 shows an example of melanocyte staining (for MART1) and endothelin 1 in UV-exposed skin and in unexposed skin. A comparison of the effects of two methods of antigen retrieval to optimize staining is shown in Fig. 4.

image

Figure 2.  Overview of immunohistochemistry.

Download figure to PowerPoint

Table 1.   Protocol for indirect immunofluorescence of melanogenic markers
For frozen sections
•Dry frozen sections for 45 min at room temperature (RT)
•Fixation with cold methanol or 4% paraformaldehyde for 20 min at 4°C (let air dry 20 min if cold methanol was used). Rinse in PBS (×3) for 5 min
•For permeabilization of cell membranes, add 0.01% Triton X for 3 min or cold methanol for 15 min at 4°C (let air dry 20 min if cold methanol was used). Rinse in PBS (×3) for 5 min. Continue with the quench or pretreat steps described below
For paraffin-embedded sections
•Deparaffinize and rehydrate paraffin-embedded slides
•Xylene or xylene substitute for 5 min (×2)
 100% EtOH for 3 min
 95% EtOH for 3 min
 70% EtOH for 3 min
 50% EtOH for 3 min
•Rinse in PBS 3 min
•Antigen retrieval (AR): antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA), 1 mM EDTA (pH 8) or 10 mM citrate buffer (pH 6). Heat in microwave oven for 10–12 min and cool down for 20 min. Rinse in PBS for 5 min
•Quench or pretreat (for peroxidase and/or biotin–streptavidin): incubate in 0.3% H2O2 in water for 30 min at RT and/or streptavidin/biotin blocking solution according to the manufacturer's instructions. Rinse in PBS for 5 min
•Mark around samples with a hydrophobic barrier pen. Rinse in PBS for 5 min
•Blocking: incubate in 1–2% BSA, 10% normal serum/PBS or Image-IT FXTM Signal Enhancer (Invitrogen Corp.) for 60 min at RT
•If Image-IT FXTM Signal Enhancer was used as a blocking reagent, rinse in PBS (×3) for 5 min and blot excess solution from sections
•Primary antibody: anti-melanocyte specific protein antibody (or antisera) diluted in 1% BSA or in 5% normal serum/PBS (see Table 3) then put 100∼300 μl antibody dilution on the sections
•Put glass slides in a humidified chamber and incubate at 4°C overnight
•Wash with 0.05% Tween-20/PBS (×4)
•Secondary antibody (conjugated): incubate in fluorescence conjugated anti-species IgG (H + L) (Molecular Probes) (1:500) in 5% normal serum/0.05% Tween-20/PBS for 60 min at RT in the dark
•Wash with 0.05% Tween-20/PBS (×3) in dark
•Counter-stain with anti-fade medium with or without DAPI and mount
Alternate approach: if the above fluorescent method is too weak, one may try the following optional steps instead of the above secondary antibody steps
•Incubate in biotinylated anti-species IgG or universal secondary antibody (1:100)/5% normal serum/PBS for 60 min at RT
•Wash in PBS (×3) for 5 min
•Incubate in fluorescein streptavidin (1:100)/5% normal serum/PBS for 60 min at RT in dark
•Wash with PBS (×3) for 5 min in dark
•Counter-stain and mount as indicated above
image

Figure 3.  Increased expression of MART1 and endothelin 1 after ultraviolet (UV) exposure. Subjects were exposed to 10 sessions of UV radiations (95% UVA and 5% UVB) in 5 weeks, total cumulative dose 4.3 kJ/m2. Samples were stained with antibodies against MART1 (green) and endothelin 1 (red). (a) Unexposed skin; (b) UV-exposed skin; (c) control (sample stained only with the secondary antibodies).

Download figure to PowerPoint

image

Figure 4.  Example of improvement of antigen specificity by optimizing antigen retrieval, blocking and antibody concentration. (a) Antigen retrieval: Vector Unmasking solution & Microwave 12 min; blocking buffer: 10% Goat Serum; antibody concentration: gp100 (αPEP13h) 1:700. (b) Antigen retrieval: 1 mM EDTA & Microwave 10 min increased signal intensity. Blocking buffer: ImageIT Signal enhancer reduced background fluorescent from second antibody; antibody concentration: gp100 (αPEP13h) 1:10 000 decreased non-specific binding of antibody.

Download figure to PowerPoint

Caveats/pitfalls

Standardization and quality control within each laboratory will minimize problems with IHC, but there are a number of important variables to keep in mind, as follows:

  • For frozen sections, fixation with 4% paraformaldehyde is stronger and provides better conservation of the shape of the cells. However, some antigens may be altered by this fixation and cold methanol should then be used.
  • The permeabilization of the membrane is more effective with triton X which allowed better staining of cytoplasmic and nuclear antigens. However, the cellular membrane could be altered by triton X and incubation time with triton X should be carefully monitored. Use of cold methanol is usually safer for skin tissues.
  • Fixation of tissue in formalin followed by antigen retrieval has been shown to provide optimal immunostaining (26). Variations in fixation are improved by microwave oven antigen retrieval (27).
  • Adhesion coating of glass slides, such as silane coating or polylysine coating, should be used to minimize section peeling.
  • The heating temperature and the time of heating seem to be the most critical events during antigen retrieval because they determine if the sections peel off the slide, fold over or provide sufficient unmasking of the antigen for optimal staining (28).
  • If a commercial antigen unmasking solution is not used, the pH value of the antigen retrieval solution for EDTA (pH 8.0) or citrate buffer (pH 6.0) is important. A report in the literature shows good results with low pH antigen unmasking solutions for some nuclear antigens (29). If a commercial antigen unmasking solution is used, it is important to shake it well before using, as prolonged storage at 4°C creates some precipitation of the buffer. It has been reported that EDTA-heat antigen retrieval is more sensitive than heat treatment with citrate (30).
  • We have found that the sensitivity of staining for melanocyte antigens is increased using heat treatment with 1 mm EDTA (Table 2). The concentration of antibodies should be less than that used with citrate heat retrieval, because EDTA antigen retrieval also increases non-specific signals if one uses the same primary antibody concentration.
  • It is important to place the slides in a humidified chamber in order to prevent the samples from drying out during the incubation with antibodies. In addition, semi-automated systems, such as the Shandon CoverplateTM Technology (Thermo Electron Corp., Waltham, MA, USA), can prevent the primary antibody from evaporating and at the same time allow for high throughput studies (31).
  • One key step in achieving a good signal-to-noise ratio is to pay special attention to making appropriate blocking steps to avoid as much non-specific antibody binding as possible. Depending on the detection system used, additional blocking steps can be employed with the traditional normal serum or bovine serum albumin (BSA), such as a blocking step with H2O2 for peroxidase, an avidin–biotin block for the avidin–biotin conjugate method, or a biotin–streptavidin block for the biotin–streptavidin method. In addition, it has been noted that incomplete paraffin removal can lead to diffuse background staining, and in such cases additional blocking steps are of little value (32).
  • To avoid experimental error caused by the cross-reactivity of antibodies, many commercially available secondary antibodies can be pre-absorbed with immunoglobulin and/or serum for other species. One should carefully check the cross-reactivities of secondary antibodies in a pilot study. For example, we have seen commercial goat anti-rabbit cross-react with mouse IgG3 antibody, even though this secondary antibody was pre-absorbed with mouse serum and IgG. This phenomenon may depend on the low frequency of IgG3 in normal mouse IgG.
  • Anti-fade reagents should be used for immunofluorescence, because the intensity of many fluorescence products is decreased by photo-bleaching. Mounting medium that includes anti-fade reagents are commercially available. They are characterized by different stabilities of the anti-fade effect, fastness and hydrophobicity. For example, the anti-fade effect of Prolong Gold (Invitrogen Corp., Carlsbad, CA, USA) remains high for several months, although one needs to wait 24 h after mounting for curing. In contrast, microscopic observations can begin immediately if Slow Fade Gold (Invitrogen Corp.) is used; however, in this case the fluorescence intensity only lasts several weeks after mounting. Another option is the product CytosealTM 60 (Richard-Allan Scientific, Kalamazoo, MI, USA), which is suitable for Q-dot and sections that have been dehydrated by ethanol and xylene.
  • The fluorescence excitation and emission wavelengths of secondary antibodies must be suitable for the fluorescent microscope filters used. Incorrect combinations of filters and fluorophores can reduce signal intensity. It is important to exclude fluorophores with overlapping emission spectra when attempting multicolour staining (The Handbook Tenth edition; Molecular Probes, Carlsbad, CA, USA).
  • It is important to always include both a positive and a negative control during each experiment to evaluate the quality of the staining and whether the protocol has been performed correctly (32). Although skin is a good positive control for melanocyte-specific proteins as it contains many diverse types of cells in the same section, tanned skin is a better positive control compared with fair skin.
Table 2.   List of melanocyte specific antibodies routinely used in our laboratory
 Optimized antibody concentration with different antigen retrieval techniques
 CloneHostName of cloneIsotypeCommercial unmasking solution (citric acid and proprietary salts)1 mM EDTA (pH 8.0)
Human MITF N-terminalMonoclonalMouseC5 + D5IgG1μg/mlμg/ml
Human MART-1 recombinantMonoclonalMouseM2-7C10 + M2-9E3IgG2bμg/ml1–2 μg/ml
TyrosinaseAntiserumRabbitαPEP7hN/A1:7001:8000
Pmel17/gp100AntiserumRabbitαPEP13hN/A1:7001:10 000
DCT/TRP2AntiserumRabbitαPEP8hN/ANot good1:4000

Many coat colour genes have been identified in mice and a wide variety of mice mutant at one or more pigment loci are available (33,34). Consequently, tissues from these mutant mice can be used not only as good positive or negative controls, but also as good subjects to investigate the role of melanocyte-specific markers. Previously, our group co-stained agouti signal protein and TYR in mouse hair follicles (35).

Sometimes there can be variable staining between samples from the same batch. We have checked to determine whether this phenomenon is caused by operator error. In our laboratory, we use an antibody that can serve as an internal control for all sections, the DNA antibody AC-30-10 (Millipore, Billerica, MA, USA). If such an internal control is not stained, one should check the fixation procedure and the storage conditions of samples. Variability in staining can also result from bad sectioning with samples of different thicknesses and/or inadequate or excessive antigen retrieval (32).

In summary, high-quality specimens, specific antibodies and optimized procedures are important essentials for successful immunohistochemical studies.

Tissue in situ hybridization

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

Immunohistochemistry is a relatively easy and reliable method to assess protein expression in the skin. However, antibodies are not always available for the target protein being studied. Therefore, it can be of interest to study the transcriptional activity of any gene by assessing its RNA expression in the skin. The TISH procedure allows for the study of mRNA levels of genes in tissues at cellular resolution. The fact that one can avoid using radioisotopes via the sensitive detection of chemically labelled nucleotides makes TISH an attractive technique. TISH has been used with success to study pigment cells in human (36–41) and in mouse (42,43) tissues. Interestingly, TISH has proven the existence of amelanotic melanocytes within the outer root sheath of senile white hair (44).

TISH for melanogenic markers

The TISH procedure can be divided into three distinct steps: (i) design of specific probes; (ii) labelling of the probes, and (iii) hybridization of the probes onto the skin samples. An overview of the TISH technique is shown in Fig. 5 and a detailed procedure for TISH is presented in Table 3. An example of melanocyte identification by TISH using a probe for TRP1 is shown in Fig. 6a. Details of buffer compositions used in TISH are shown in Table 4. The sequences of several melanogenic probes that we have designed and tried successfully are listed in Table 5.

image

Figure 5.  Overview of TISH.

Download figure to PowerPoint

Table 3.   Protocol for tissue in situ hybridization of melanogenic markers
Design of probes
Oligonucleotide probes specific for the gene in question need to be designed first. Target sites are selected based on the analysis of sequence matches and mismatches, BLAST (GenBank). Designed probes should not show evidence of cross-reaction with sequences of other genes. Complementary DNA of a gene of interest is cloned into a plasmid that contains both Sp6 and T7 promoters on each side of the insertion site
Labelling of probes
The probes have to be tailed with digoxigenin-11-dUTP, e.g. DIG RNA labelling kit (Roche, Basel, Switzerland)
•Take 20–30 ng of DNA template (for 500 bp PCR product)
•Add 2 μl dNTP mix (mix of dATP, dCTP, dGTP, dUTP, DIG-11-UTP), 2 μl 10X transcription buffer, 1 μl RNAse inhibitor, 2 μl RNA polymerase (T7 or SP6, respectively, for antisense and sense probes), and nuclease free water for a total of 21 μl
•Incubate at 37°C for 2 h, then add 2 μl DNAse and incubate again for 15 min at 37°C
  After purification, the RNA probes can be stored at −20°C for 1 year.
Hybridization
•For combination with immunohistochemistry, the samples have to be deparaffinized and rehydrated. However, xylene should be used three times and 5 min each, and then ethanol 100% four times 5 min each and finally a decreasing concentration of ethanol (90, 80, 70 and 50%) for 5 min each. Finally rinse with PBS for 10 min
•Put the slides in 2 ml Ag retrieval solution to 198 ml ddH2O. Heat in microwave for 12 min and cool for 20 min
•Circle the skin samples with a hydrophobic pen
•Wash slides in glycine solution (2 mg/ml in PBS) for 10 min with gentle shaking. Then wash in PBS (2×) for 3 min
•Put slides in 200 ml acetylation buffer, and add 500 μl acetic anhydride. Incubate at RT for 15 min with continuous agitation
•Wash twice in 4X SSC for 10 min also with continuous agitation
•Incubate in pre-warmed pre-hybridization solution (in a water bath at 47°C) for more than 30 min (45–60 min is optimal)
•During this time, prepare the probe mixture. 5 μl of probes and 2 μl of RNAmix are needed for each slide. First, mix and denature the probes at 65°C for 5 min. Then, cool the probe mixture on ice more than 5 min
  If there is a lot of waiting time until the probes are used, leave them on ice to remain denatured.
•Add to the probes and RNAmix, 0.5 μl 10% NTS, 0.5 μl 10% SDS and 80 μl hybridization buffer and mix. Spin down to get rid of bubbles and apply 80 μl of the prepared probe mixture per slide. Cover with cover glass and incubate in a humid hybridization tray overnight at 47°C
•The following day, prepare three jars of pre-hybridization solution and leave them in a water-bath for 20–30 min at 47°C. Once the solution is pre-warmed, drain away the probe, dip the slides for a quick wash in pre-hybridization solution and carefully remove the cover glasses. Then, wash the samples (3×) for 20 min in a water bath (47°C) with the jars of pre-hybridization solution already warmed-up
•Wash samples in pre-warmed NTE buffer for 5 min (water bath at 37°C) and after keep the buffer. Treat the samples with pre-warmed RNase A solution for 30 min in water bath at 37°C, then return the samples into NTE buffer and incubate for 3 min in a water bath at 37°C. Finally, wash the samples with TBS for 1 min at RT
•Cover the samples with blocking solution [5% blocking agent (Roche) in TBS] and incubate for more than 30 min in a humidified chamber. Wipe off all surrounding liquid before adding blocking solution
•Wash with TBS for 5 min with gentle shaking. Remove the slides and wipe off all surrounding liquid. Treat the samples with anti-DIG-HRP conjugate, 1:300 dilution in TBS (80 μl per sample). Incubate for 45 min in a wet box at RT
•Wash the samples in TBST (2×) for 3 min with smooth shaking. Add one to two drops of Biotinyl tyramide to the samples, and incubate at RT for 15 min precisely
•Wash the samples with TBST (3×) for 3 min with gentle shaking. Add one to two drops of secondary avidin-HRP on the samples, and incubate at RT for 15 min precisely
•Wash the samples with TBST (3×) for 3 min with gentle shaking then with TBS two times for 3 min with gentle shaking
•Wipe off all surrounding liquid and cover the samples with 15 μl of VIP substrate solution at RT for 5 min
•Use ddH2O to stop the reaction. Four to five quick washes then wash (3×) for 2 min with gentle shaking
Wipe off all surrounding liquid and add mounting medium
image

Figure 6.  Examples of TISH staining. (a) TISH with probes against TYRP1: (left) antisense probes, (right) sense probes. (b) Combination of TISH and IHC: (left) TISH with probes against Sox10, (right) TISH with probes against Sox10 combined with IHC with antibody against MART1. Note that the TISH reveals the presence of Sox10 RNA both in melanocytes and in keratinocytes.

Download figure to PowerPoint

Table 4.   Buffers used for TISH
Dionized formamide
 Add 30 ml of resin + 105 ml of DNAse free formamide
Pre-hybridization buffer
 1:1 of 4X SSC (made with DEPC water) and formamide
Acetylation buffer
 TEA (triethylamine) 2.67 ml + DEPC water 144 ml + HCL 0.75 ml + DEPC water 32.58 ml Please follow the same order
Hybridization buffer
 1 m Tris–HCl, pH 7.4, 0.95 ml + 0.5 m EDTA, pH 8.0, 0.1 ml + 5 m NaCl 2.4 ml + formamide 23.8 ml + 50% dextran sulphate 9.52 ml + 50x Denhardt's solution 0.95 ml + DEPC H2O 2.28 ml for a total of 40 ml
RNA mix = salmon sperm DNA + ribonucleic acid + yeast tRNA
 For 80 μl of RNA mix: salmon sperm DNA 20 μl, ribonucleic acid 25.04 μl, yeast tRNA 20 μl, H2O 15 μl
NTE buffer = RNase A washing buffer
 For 360 ml: H2O 324 ml, 5 m NaCl 36 ml, 1 m Tris–HCl pH 8.0 36 ml, 0.5 m EDTA 0.18 ml
Rnase A solution = NTE buffer (see above) 180 ml + ribonuclease A (20 μg/ml)
Table 5.   Sequence of some riboprobes used for TISH experiments in the skin
Human TRP1 (X51420)
 HTRP1-T7 (1591–1612) GCGCGTAATACGACTCACTATAGGG-CAAAAATGAGTGCAACCAGTAA
 HTRP1-T3 (1091–1110) CGCGCAATTAACCCTCACTAAAGG-GACCAATGGTGCAACGTCTT
Human tyrosinase (M27160)
 HTyrosinase-T7 (1411–1432) GCGCGTAATACGACTCACTATAGGG-TGGGGTTCTGGATTTGTCATGG
 HTyrosinase-T3 (911–930) CGCGCAATTAACCCTCACTAAAGG-TACCTCACTTTAGCAAAGCA
Human DCT (NM_001922)
 HDCT-T7 (1101–1122) GCGCGTAATACGACTCACTATAGGG-GCCAATGAGTCGCTGGAGATCT
 HDCT-SP6 (601–620) CATACGATTTAGGTGACACTATAG-GAGGTGCGAGCCGACACAAG
Human GP100 (NM_006928)
 HGP100-3 T7 (1801–1822) GCGCGTAATACGACTCACTATAGGG-CGGAACCTGCCCAAGGCCTGCT
 HGP100-3 T3 (1301–1320) CGCGCAATTAACCCTCACTAAAGG-GTGGAGACCACAGCTAGAGA
Human SOX10 (NM_006941)
 SOX10T7 (861–882) GCGCGTAATACGACTCACTATAGGG-TGCCTTGCCCGACTGCAGTTCT
 SOX10SP6 (661–680) CATACGATTTAGGTGACACTATAG-GGGAAGGCCGCCCAGGGCGA

Caveats/pitfalls

  • DNA, RNA and oligonucleotide probes can be used to perform TISH. However, RNA probes usually perform better because of their higher sensitivity.
  • The length of probes is also an important consideration. Whereas some groups have used cDNAs of more than 1 kb (45), we have obtained better results with cDNAs of ∼500 bp (18,46).
  • Typically, several different probes for each gene/protein of interest are designed and then tested to screen for the best ones. Sense probes also need to be designed to serve as negative controls. A competitive hybridization can be performed using cold probes to serve as a negative control.
  • A high fidelity enzyme should be used to perform the PCR.
  • Frozen tissues or paraffin-embedded tissues can be used for TISH. However, we usually work with paraffin tissues as they are easier to store.
  • All instruments should be washed with ddH2O and treated with RNAse before use. Every phase during pre-hybridization and hybridization must be done in RNAse-free conditions. In addition, it is very important to prevent samples from drying completely.
  • Similar to IHC, samples are placed in a humidified chamber to prevent them from drying out during the overnight incubation with the probes. Moreover, bubbles should be avoided when applying the probe mixture to samples as false negative or heterogeneous staining could result.
  • We generally use the tyramide signal amplification reaction, but any signal amplification system designed for IHC can be used for chemical detection in TISH. However, we avoid DAB staining as the brownish colour can be difficult to distinguish from melanin. Fluorescent staining can also be used.
  • When the chemical reactants are added to samples, it is very important to incubate all samples exactly for the same time as longer incubation can lead to further increases in signal intensity. It is possible to monitor the change in colour using a microscope after adding the VIP substrate, however, the stain should not be developed for longer than 10 min.
  • Finally, carefully dry the samples before adding the mounting medium as remaining water can strongly decrease the signal over the next 12–24 h.

Concluding remarks

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

The advent of an increased number of reliable melanocyte-specific antibodies, improved TISH techniques, powerful microscopes and superior software has allowed for the identification and analysis of melanocytes in the skin under in vivo conditions. Immunohistochemistry is an easy and reliable technique. It allows the analysis of protein expressions in vivo conditions and it is generally our first choice to study a protein of interest. TISH technique is more complicated to perform and requires the design and the production of specific probes. However, it allows the study of the mRNA expression and is very useful in complement of the IHC or when good antibodies are not available. Both of these techniques can be combined to identify melanocytes, e.g. with a melanocyte marker antibody (such as TYR or MART1) after performing TISH with probes against a gene expressed by keratinocytes and/or melanocytes. In such a case, the experiment must be stopped before fixation and then standard IHC can follow as described above, starting with the primary antibody incubation (Fig. 6b). The combination of IHC and TISH also allows for the simultaneous evaluation of RNA and protein levels of one gene of interest. Hopefully, in the coming years, in vivo techniques, such as IHC, TISH, and DNA microarray analysis after laser microdissection, will provide crucial data on the relationships between all types of cells in the skin. Such information will help us to better understand the processes leading to pigmentation and/or to carcinogenesis.

Acknowledgements

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.

References

  1. Top of page
  2. Abstract:
  3. Introduction
  4. Interfollicular and follicular melanocyte markers
  5. Immunohistochemistry
  6. Tissue in situ hybridization
  7. Concluding remarks
  8. Acknowledgements
  9. References
  • 1
    Luger T A, Scholzen T, Brzoska T, Becher E, Slominski A, Paus R. Cutaneous immunomodulation and coordination of skin stress responses by alpha-melanocyte-stimulating hormone. Ann N Y Acad Sci 1998: 840: 381394.
  • 2
    Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Res 2004: 17: 96110.
  • 3
    Yamaguchi Y, Hearing V J, Itami S, Yoshikawa K, Katayama I. Mesenchymal–epithelial interactions in the skin: aiming for site-specific tissue regeneration. J Dermatol Sci 2005: 40: 19.
  • 4
    Yamaguchi Y, Itami S, Watabe H et al. Mesenchymal-epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation. J Cell Biol 2004: 165: 275285.
  • 5
    Westerhof W. The discovery of the human melanocyte. Pigment Cell Res 2006: 19: 183193.
  • 6
    Fitzpatrick T B, Becker S W Jr, Lerner A B, Montgomery H. Tyrosinase in human skin: demonstration of its presence and of its role in human melanin formation. Science 1950: 112: 223225.
  • 7
    Lerner A B, Fitzpatrick T B. Biochemistry of melanin formation. Physiol Rev 1950: 30: 91126.
  • 8
    Hearing V J, Ekel T M. Mammalian tyrosinase: a comparison of tyrosine hydroxylation and melanin formation. Biochem J 1976: 157: 549557.
  • 9
    King R A, Olds D P, Witkop Jr C J. Characterization of human hairbulb tyrosinase: properties of normal and albino enzyme. J Invest Dermatol 1978: 71: 136139.
  • 10
    Szabo G. The number of melanocytes in human epidermis. Br Med J 1954: 1: 10161017.
  • 11
    Bertolotto C, Abbe P, Hemesath T J et al. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes. J Cell Biol 1998: 142: 827835.
  • 12
    Shibahara S, Yasumoto K, Amae S et al. Regulation of pigment cell specific gene expression by MITF. Pigment Cell Res 2000: 13: 98102.
  • 13
    Levy C, Khaled M, Fisher D E. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med 2006: 12: 406414.
  • 14
    Korner A M, Pawelek J. Dopachrome conversion: a possible control point in melanin biosynthesis. J Invest Dermatol 1980: 75: 192195.
  • 15
    Steel K P, Davidson D R, Jackson I J. TRP2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 1992: 115: 11111119.
  • 16
    Kawakami Y, Robbins P F, Wang R F, Parkhurst M R, Kang X, Rosenberg S A. Tumor antigens recognized by T cells: the use of melanosomal proteins in the immunotherapy of melanoma. J Immunother 1998: 21: 237246.
  • 17
    Hoashi T, Watabe H, Muller J, Yamaguchi Y, Vieira W D, Hearing V J. MART-1 is required for the function of the melanosomal matrix protein Pmel17/gp100 and the maturation of melanosomes. J Biol Chem 2005: 280: 1400614016.
  • 18
    Valencia J C, Watabe H, Chi A et al. Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes. J Cell Sci 2006: 119: 10801091.
  • 19
    Nishimura E K, Jordan S A, Oshima H et al. Dominant role of the niche in melanocyte stem-cell fate determination. Nature 2002: 416: 854860.
  • 20
    Grichnik J M, Crawford J, Jimenez F et al. Human recombinant stem-cell factor induces melanocytic hyperplasia in susceptible patients. J Am Acad Dermatol 1995: 33: 577583.
  • 21
    Horikawa T, Norris D A, Johnson T W et al. DOPA-negative melanocytes in the outer root sheath of human hair follicles express premelanosomal antigens but not a melanosomal antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1, and TRP-2. J Invest Dermatol 1996: 106: 2835.
  • 22
    Tobin D J, Bystryn J C. Different populations of melanocytes are present in hair follicles and epidermis. Pigment Cell Res 1996: 9: 304310.
  • 23
    Commo S, Bernard B A. Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study. Pigment Cell Res 2000: 13: 253259.
  • 24
    Commo S, Gaillard O, Thibaut S, Bernard B A. Absence of TRP-2 in melanogenic melanocytes of human hair. Pigment Cell Res 2004: 17: 488497.
  • 25
    Lan C C, Wu C S, Cheng C M, Yu C L, Chen G S, Yu H S. Pigmentation in basal cell carcinoma involves enhanced endothelin-1 expression. Exp Dermatol 2005: 14: 528534.
  • 26
    Prento P, Lyon H. Commercial formalin substitutes for histopathology. Biotech Histochem 1997: 72: 273282.
  • 27
    Williams J H, Mepham B L, Wright D H. Tissue preparation for immunocytochemistry. J Clin Pathol 1997: 50: 422428.
  • 28
    Shi S R, Cote R J, Taylor C R. Antigen retrieval techniques: current perspectives. J Histochem Cytochem 2001: 49: 931937.
  • 29
    Shi S R, Imam S A, Young L, Cote R J, Taylor C R. Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 1995: 43: 193201.
  • 30
    Pileri S A, Roncador G, Ceccarelli C et al. Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 1997: 183: 116123.
  • 31
    Douwes Dekker P B, Boon M E, Vardaxis N J. Microwave immunoincubations using coverplate units. Eur J Morphol 1993: 31: 298308.
  • 32
    Taylor C R. The total test approach to standardization of immunohistochemistry. Arch Pathol Lab Med 2000: 124: 945951.
  • 33
    Sturm R A, Teasdale R D, Box N F. Human pigmentation genes: identification, structure and consequences of polymorphic variation. Gene 2001: 277: 4962.
  • 34
    Bennett D C, Lamoreux M L. The color loci of mice – a genetic century. Pigment Cell Res 2003: 16: 333344.
  • 35
    Matsunaga N, Virador V, Santis C et al. In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody. Biochem Biophys Res Commun 2000: 270: 176182.
  • 36
    Scott G, Stoler M, Sarkar S, Halaban R. Localization of basic fibroblast growth factor mRNA in melanocytic lesions by in situ hybridization. J Invest Dermatol 1991: 96: 318322.
  • 37
    Fleming M G, Howe S F, Graf Jr L H. Expression of insulin-like growth factor I (IGF-I) in nevi and melanomas. Am J Dermatopathol 1994: 16: 383391.
  • 38
    Ciotti P, Pesce G P, Cafiero F et al. Intercellular adhesion molecule-1 (ICAM-1) and granulocyte-macrophage colony stimulating factor (GM-CSF) co-expression in cutaneous malignant melanoma lesions. Melanoma Res 1999: 9: 253260.
  • 39
    Duncan L M, Deeds J, Cronin F E et al. Melastatin expression and prognosis in cutaneous malignant melanoma. J Clin Oncol 2001: 19: 568576.
  • 40
    Suzuki I, Kato T, Motokawa T, Tomita Y, Nakamura E, Katagiri T. Increase of pro-opiomelanocortin mRNA prior to tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, Pmel-17/gp100, and P-protein mRNA in human skin after ultraviolet B irradiation. J Invest Dermatol 2002: 118: 7378.
  • 41
    Motokawa T, Kato T, Katagiri T et al. Messenger RNA levels of melanogenesis-associated genes in lentigo senilis lesions. J Dermatol Sci 2005: 37: 120123.
  • 42
    Potterf S B, Mollaaghababa R, Hou L et al. Analysis of SOX10 function in neural crest-derived melanocyte development: SOX10-dependent transcriptional control of dopachrome tautomerase. Dev Biol 2001: 237: 245257.
  • 43
    Watanabe K, Takeda K, Yasumoto K et al. Identification of a distal enhancer for the melanocyte-specific promoter of the MITF gene. Pigment Cell Res 2002: 15: 201211.
  • 44
    Takada K, Sugiyama K, Yamamoto I, Oba K, Takeuchi T. Presence of amelanotic melanocytes within the outer root sheath in senile white hair. J Invest Dermatol 1992: 99: 629633.
  • 45
    Suzuki I, Motokawa T. In situ hybridization: an informative technique for pigment cell researchers. Pigment Cell Res 2004: 17: 1014.
  • 46
    Takahashi K, Hoashi T, Yamaguchi Y et al. UV increases the nuclear localization of apurinic/apyrimidinic endonuclease/redox effector factor-1 in human skin. J Invest Dermatol 2006: 126: 27232726.