Application of the fractionator and vertical slices to estimate total capillary length in skeletal muscle

A new stereological method is proposed which combines vertical slice projec tions with the fractionator to estimate the total capillary length in a ske letal muscle. The method was demonstrated on the soleus muscle of a Wistar rat. The implementation required capillary highlighting, tissue sampling, a nd data acquisition in the form of intersection counts between capillary pr ojections and cycloid test lines. The capillaries were demonstrated using v ascular perfusion (with gelatine) of the hind leg of the rat. The sampling procedure followed the fractionator design, namely a multistage systematic sampling design with a known sampling fraction at each stage. To make the d esign unbiased, vertical slices were used; for efficiency, the vertical axi s was chosen parallel to the main axis of the muscle. As prescribed to avoi d bias, the cycloid test lines were superimposed on the slice projections, viewed under the light microscope, with their minor axes normal to the vert ical axis. The estimation precision was compared for different sampling and subsampling fractions. The proposed method was globally highly efficient, unbiased, and easy to implement.