Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts

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Colocalization of Glial Fibrillary Acidic Protein, Metallothionein, and MHC II in Human, Rat, NOD/SCID, and Nude Mouse Skin Keratinocytes and Fibroblasts Lusine Danielyan, Genrich Tolstonog, Peter Traub, Juergen Salvetter, Christoph H. Gleiter, Dieter Reisig, Rolf Gebhardt, Gayane H. Buniatian Journal of Investigative Dermatology Volume 127, Issue 3, Pages 555-563 (March 2007) DOI: 10.1038/sj.jid.5700575 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Astrocyte-associated antigens in (a–i) frozen young (10 years) and (j, k) adult human skin sections. (a) Expression of GFAP detected using mouse mAb GFAP. (b) Expression of GFAP detected using rabbit pAb GFAP. (c) Double staining for GFAP (green) and desmin (red). (d) Staining for desmin alone. (e) Double staining for GFAP (green) and vimentin (red). (f) Double staining for GFAP (green) and SMAA (red). (g) Staining for SMAA alone. (h) Double staining for GFAP (green) and SMAA (red). (i) Double staining for GFAP (green) and MT (red). (j) Double staining for GFAP (green) and MHC II (red). (k) Staining for GFAP (green). Blue arrows point to stratum corneum, white arrows to stratum basalis, yellow arrows to fibroblasts adjacent to stratum basalis, red arrows to papilla structures, and magenta arrows to fibroblasts in the lower layers of dermis. Yellow arrowheads to sweat glands, white arrowheads to vessels, and red arrowheads to hair follicles. Cy3-conjugated anti-mouse IgG from goat was used for the detection of mouse mAb GFAP, mAb desmin, mAb vimentin, mAb SMAA, and mAb MT-labelling sites. Skin structures labelled with rabbit pAb GFAP were detected using FITC-conjugated anti-rabbit IgG from goat. The preservation of skin structures and cells which could not be detected by distinct antibodies was ascertained by counterstaining of cell nuclei by DAPI (blue). Bar: in (a, e, f, h, i)=10μm; in (b–d, g, j, k)=20μm. Journal of Investigative Dermatology 2007 127, 555-563DOI: (10.1038/sj.jid.5700575) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Differentiation of human keratinocytes and fibroblasts in culture. (a–c) Double labelling of 7-day-old keratinocytes with pAb GFAP and mAb MT: (a) merge of GFAP and MT staining; (b) staining for GFAP; and (c) staining for MT. (d–f) Double labelling of 2-day-old fibroblasts with pAb GFAP and mAb desmin: (d) merge of GFAP and desmin staining; (e) staining for GFAP; and (f) staining for desmin. (g–i) Double labelling of 7-day-old fibroblasts with pAb GFAP and mAb MT: (g) merge of GFAP and desmin staining; (h) staining for GFAP; and (i) staining for desmin. The secondary antibody used in (b, e, h) was rabbit antiserum FITC-conjugated anti-rabbit IgG from goat, whereas in (c, f, i) was Cy3-conjugated anti-mouse IgG from goat. Bar=20μm. Journal of Investigative Dermatology 2007 127, 555-563DOI: (10.1038/sj.jid.5700575) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Astrocyte-associated antigens in frozen rat skin sections. (a) Labelling with pAb GFAP. (b) Labelling with mAb SMAA (red). (c) Double labelling with pAb GFAP (green) and mAb SMAA (red). (d) Labelling with mAb MHC II. (e) Double labelling with pAb GFAP (green) and mAb desmin (red). (f) Double labelling with pAb GFAP (green) and mAb vimentin. Skin structures labelled with anti-GFAP were detected using FITC-conjugated anti-rabbit IgG from goat, whereas Cy3-conjugated anti-mouse IgG from goat was used for detection of mAb desmin, mAb vimentin, mAb SMAA, and mAb MHC II. Blue arrows point to epidermis; yellow arrows to fibroblasts (F); white arrows to follicle cortex (Co); red arrows to cuticle (Cu); and magenta arrows to outer sheet of the hair follicles. Bar: in (a–d, f)=10μm; in (e)=20μm. Journal of Investigative Dermatology 2007 127, 555-563DOI: (10.1038/sj.jid.5700575) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Colocalization of GFAP and MHC II in normal and mutant murine skin. (a–c) Double staining of CD1 Balb C mouse skin sections for GFAP and MHC. (a) Merged micrograph of GFAP and MHC immune reaction; (b) staining for GFAP; and (c) staining for MHC II. (d–f) Double staining of C75BL/6 mouse skin sections for GFAP and MHC II. (d) Merged micrograph of GFAP and MHC immune reaction; (e) staining for GFAP; and (f) staining for MHC II. (g–i) Double staining of CD1-Foxn1 nu nude mouse skin sections for GFAP and MHC. (g) Merged micrograph of GFAP and MHC immune reaction; (h) staining for GFAP; and (i) staining for MHC II. (j–l) Double staining of NOD-SCID mouse skin sections for GFAP and MHC. (j) Merged micrograph, staining for GFAP and MHC immune reaction; (k) staining for GFAP; and (l) staining for MHC II. Skin structures labelled with anti-GFAP were detected using FITC-conjugated anti-rabbit IgG from goat, whereas Cy3-conjugated anti-mouse IgG from goat was used for detection of mAb MHC II. Because MHC II was very strongly expressed in (a, c) CD1 BalbC, (d, f) C57BL/6, and in (g, i) nude mice, the images illustrating immunoreaction of MHC II had to be taken at 10-fold faster exposure (less sensitivity) than those of GFAP, whereas in (j, l) NOD/SCID mice exposure times were equal. Blue arrows point to epidermis; yellow arrows to dermal fibroblasts; and white arrows to follicles. Bar=10μm. Journal of Investigative Dermatology 2007 127, 555-563DOI: (10.1038/sj.jid.5700575) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The authenticity of GFAP expression in skin. (a–d) CLS microscopy of skin preparations stained for GFAP. (a) Labelling of newborn rat skin sections with pAb GFAP. (b) Phase contrast micrograph of the field shown in (a). (c) Cultured human fibroblasts stained for GFAP. (d) Labelling of newborn rat lung section with pAb GFAP. GFAP was detected using rabbit antiserum and FITC-conjugated anti-rabbit IgG from goat. Blue arrows point to epidermis; yellow arrows to fibroblasts (f); white arrows to follicle cortex (Co); red arrows to cuticle (Cu); and magenta arrows to outer sheet of the hair follicles. (e) Control widefield immunofluorescence assay: human skin section treated with mixture of FITC-conjugated anti-rabbit IgG from goat and Cy3-conjugated anti-mouse IgG from goat. Bar: in (a, b)=10μm; in (c–e,)=20μm. (f, g) Immunoblot analysis of GFAP. (f) Immunoblot analysis of GFAP in homogenates of newborn rat skin (lane 1, 34.5μg; lane 2, 3.0μg; and lane 3, 1.5μg); (g) immunoblot analysis of GFAP in homogenates of T6 cells (lane 1, 12.5μg) and newborn rat astroglial culture (lane 2, 21.5μg). In all tested tissues, mAb GFAP labelled two major protein bands with apparent molecular masses of ∼55 and ∼52kDa. Journal of Investigative Dermatology 2007 127, 555-563DOI: (10.1038/sj.jid.5700575) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions