Dual-channel laser scanning microscopy for the identification and quantification of proliferating skeletal muscle satellite cells following synergist ablation.

Proliferating skeletal muscle satellite cells are the source of additional myonuclei which allow skeletal muscle to grow and regenerate. Previously, proliferating satellite cells were identified in situ by techniques which were limited either by tissue processing time or inability to observe complete muscle sections, or by errors made in separating these cells from proliferating nonmyogenic cells. To overcome these problems a new method has been devised for the identification and quantification of proliferating satellite cells in situ by light microscopy. The technique involves dual-channel laser scanning imaging of whole muscle sections for the localisation of both the muscle fibre basal lamina and the cell division marker bromodeoxyuridine. Using this technique satellite cell proliferation was quantified in mouse limb muscle following synergist ablation. Dual-channel laser scanning microscopy allowed precise localisation of proliferating satellite cells in the experimental model and, after 4 d, synergist ablation was shown to have produced significant satellite cell proliferation when compared with contralateral and sham-operated controls.