Label-Retaining Cells (Presumptive Stem Cells) of Mice Vibrissae Do Not Express Gap Junction Protein Connexin 43  Maja Matic, Marcia Simon  Journal of Investigative Dermatology Symposium Proceedings  Volume 8, Issue 1, Pages 91-95 (June 2003) DOI: 10.1046/j.1523-1747.2003.12179.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 LRC and Cx43 expression. Sections of the bulge area of vibrissal (A,B), and pelage follicles (C,D). (A,C) bright field micrographs; (B,D) fluorescent micrographs. (B′) is a diagram of (A,B). The focal plane was adjusted to visualize either Cx43 (B,D), or [3H]thymidine (A,C). The section in (B) was photographed through a green filter to allow some background autofluorescence to be visible for orientation purposes. Arrows point to the cells that express Cx43. Note that LRC do not express Cx43. Scale bar: 12 μm. Journal of Investigative Dermatology Symposium Proceedings 2003 8, 91-95DOI: (10.1046/j.1523-1747.2003.12179.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 LRC of a mouse vibrissal follicle. Fluorescent micrographs of a longitudinal section of mouse vibrissal follicle. (A) A low magnification view of vibrissal follicle. b, bulb; bg, bulge; dp, dermal papilla; s, sebaceous gland. The area within the stars is magnified in (B). (B) A high magnification view of LRC and Cx43 expressing cells. Cx43 plaque is marked by the white arrow. A white arrowhead points to a LRC. Note that LRC do not express Cx43. Yellow staining probably originates from lipids. (B′) A diagram of three cells that express Cx43 shown in (B). The black arrow depicts the Cx43 plaque that corresponds to the plaque pointed by the white arrow in (B). Scale bar: (A) 96 μm; (B) 4.8 μm. Journal of Investigative Dermatology Symposium Proceedings 2003 8, 91-95DOI: (10.1046/j.1523-1747.2003.12179.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cx43 expression in human neonatal foreskin. Fluorescent micrographs of two different regions of the epidermis (A,B) and (C). Cx43 (FITC) expression (A,B). Yellow background in the basal cells in (A) is a “bleed through” the green filter of rhodamine-stained K19. The large arrow in (A), points to the cell that apparently lacks Cx43 expression. The small arrows point to the Cx43 puncta on the neighboring cells in the basal layer. Note the high density of Cx43 puncta on the cells in the suprabasal layers (B). Double fluorescence, K19 (rhodamine) and Cx43 (FITC) expression (C). The arrows in (C) point to the cells with no apparent Cx43 expression. Scale bar: 4.8 μm. Journal of Investigative Dermatology Symposium Proceedings 2003 8, 91-95DOI: (10.1046/j.1523-1747.2003.12179.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cx43 expression in the bulge region of the human hair follicle. Fluorescent micrographs. Low magnification view (A,B). The rectangle marks the region viewed under high magnification (C,D). Cx43 (FITC) expression (A,C). K19 (rhodamine) expression (B). Double fluorescence, Cx43 (FITC) and K19 (rhodamine) expression in the bulge (D). Arrows point to the cells in the basal layer of the ORS with no apparent expression of Cx43 (C). Inner root sheath in (C) is overexposed in order for Cx43-free cells to be visible due to “bleed through” of rhodamine-stained K19. Arrowheads in (D) point to the Cx43 puncta in cells adjacent to those that are Cx43 negative. s, sebaceous gland. Scale bar: (A,B) 96 μm; (C,D) 12 μm. Journal of Investigative Dermatology Symposium Proceedings 2003 8, 91-95DOI: (10.1046/j.1523-1747.2003.12179.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Flow cytometry analysis. Dot plot in arbitrary units on a linear scale showing FSC and SSC (A). Each dot represents the FSC and SSC values for a single cell. An oval gate is used to exclude cell debris and select cells for dual fluorescent analysis. Dot plot showing fluorescent intensity of keratinocytes double labeled with antibody against Cx43 (R-PE), y-axis, and antibody against K14 (FITC), x-axis, measured in arbitrary units on a log scale (B). Quadrants are established using isotype controls, and single color positive controls. Suprabasal cells (Cx43+, K14–) are located in the upper left quadrant, R4. Presumptive stem cells (Cx43–, K14+) are located in the lower right quadrant, R2, whereas the rest of the basal cells (Cx43+, K14+) are located in the upper right quadrant, R3. Cells with nonspecific binding are located in the lower left quadrant, R0, and likely present nonkeratinocyte population. Dot plots showing FSC and SSC values (C–E) for presumptive stem cells (C); basal cells, excluding presumptive stem cells (D); and suprabasal cells (E). Data are representative of five separate experiments. Journal of Investigative Dermatology Symposium Proceedings 2003 8, 91-95DOI: (10.1046/j.1523-1747.2003.12179.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions