Figure 1.

Experimental approach for separate analysis of attacked and nonattacked myofiber subsets in sIBM. A: The focal nature of inflammatory infiltrates in IBM. Muscle tissue was stained with anti-CD8 (red) and anti-HLA-ABC (green) antibodies, as described in Materials and Methods. A shows a confocal image, C shows unembedded dry tissue. All myofibers are HLA-ABC positive, but only some are attacked by CD8⁺ T cells (A_IBM), whereas others are spared (N_IBM). B: Myofibers in direct contact with at least three CD8⁺ T cells or invaded by at least one CD8⁺ T cell were defined as A_IBM. Myofibers not in contact with any CD8⁺ T cells were defined as N_IBM. Ambiguous myofibers were not sampled. Laser-capture microdissection was used to pressure catapult the different types of myofibers into collecting tubes. In total, 100,000 μm² of A_IBM and N_IBM myofibers was sampled from each patient with sIBM, as was the same amount of H_CTRL myofibers from each control subject. C: Double immunostaining for HLA-ABC (green) and CD8 (red), according to the protocol described in Materials and Methods. Only myofiber tissue (star) was isolated, avoiding surrounding lymphocytes (red). D: Corresponding bright-light image after the myofiber was dissected and catapulted out of the tissue. C and D: The numbers in the yellow fields refer to apparatus parameters. Scale bars = 50 μm (A and C).