Journal: The Journal of Cell Biology
Article Title: ?3?1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane
Figure Lengend Snippet: Distributions of α6β4 and laminin-5 in the developing skin of normal and α3-null embryos. Frozen sections from mouse embryonic skin at days E15.5 ( A–F ) or E17.5 ( G–L ) of development were stained by double-label immunofluorescence with either monoclonal antibody 346-11A against the β4 integrin subunit ( A , C , and G ) or GoH3 monoclonal antibody against the α6 integrin subunit ( E , I , and K ), and anti–laminin-5 serum ( B , D , H , and F , J , L , respectively). Control sections were from wild-type embryos ( A–D ) or heterozygous embryos ( G and H ). In wild-type E15.5 embryos, α6β4 and laminin-5 codistributed to the basement membrane zone in more stratified regions ( C and D ), but not in less stratified regions ( A and B ); the width of the epidermis in each panel is indicated by a double-headed arrow. In α3-null embryos at E15.5 ( E and F ) and E17.5 ( I and J ), arrowheads point to areas of laminin-5 staining in areas of disorganized basement membrane, below the α6-positive basal keratinocytes; the skin in E and F is folded back on itself. ( K and L ) Higher magnification of α3-null skin at E17.5 showing α6-negative, basal keratinocytes that have separated from the laminin-5 positive basement membrane, marked by arrowheads. e , epidermis; d , dermis. Bars: (shown in J for A–J ) 50 μm; and (in L for K and L ) 50 μm.
Article Snippet: Preparation of Laminin-5–rich Extracellular Matrix from Keratinocytes To prepare laminin-5–rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described ( ) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:3 mix of Ham's F12 and DMEM), 1.8 × 10−4 M adenine, 10% FBS, HICE mix, 100 U/ml penicillin, and 100 μg/ml streptomycin) or serum-free Keratinocyte Growth Medium ( GIBCO BRL ) supplemented with EGF and bovine pituitary extract, as directed by the manufacturer.
Techniques: Staining, Immunofluorescence