Localization of laminin-5 in the epidermal basement membrane

Laminin-5 (kalinin/nicein/BM600) is a component of the epidermal basement membrane zone. Previous en bloc pre-embedding immunogold electron microscopy (EM) has shown that the epitope of GB3, a monoclonal antibody against laminin-5, is present in the lamina lucida (LL). However, precise localization of the entire laminin-5 molecule was unclear because of uneven and limited penetration of gold-labeled antibody in pre-embedding immunolabeling. In addition, the location of the GB3 epitope may not directly represent the location of the laminin-5 moelcule itself. To elucidate the precise ultrastructural distribution of the entire laminin-5 molecule, we used polyclonal antibodies against different sites of laminin-5. Dual staining immunofluorescence with anti-laminin-5 and anti-melanocyte antibodies and immunoperoxidase EM showed that laminin-5 was present only beneath keratinocytes and not beneath melanocytes. Both cryoultramicrotomy and postembedding immunogold EM demonstrated that laminin-5 was localized to the lamina densa (LD) and the lower LL, with major labeling beneath the hemidesmosome. Quantitative analysis showed that 67-69% of gold particles were distributed to the LD and 88-90% were distributed beneath the hemidesmosome. Our results indicate that laminin-5 is localized mainly to the LD and partially to the lower LL, and is associated predominantly with hemidesmosomes.