AUTOMATED MUSCLE FIBER TYPE POPULATION ANALYSIS WITH IMAGEJ OF WHOLE RAT MUSCLES USING RAPID MYOSIN HEAVY CHAIN IMMUNOHISTOCHEMISTRY

Introduction: Skeletal muscle consists of different fiber types which adapt to exercise, aging, disease, or trauma. Here we present a protocol for fast staining, automatic acquisition, and quantification of fiber populations with ImageJ. Methods: Biceps and lumbrical muscles were harvested from Sprague-Dawley rats. Quadruple immunohistochemical staining was performed on single sections using antibodies against myosin heavy chains and secondary fluorescent antibodies. Slides were scanned automatically with a slide scanner. Manual and automatic analyses were performed and compared statistically. Results: The protocol provided rapid and reliable staining for automated image acquisition. Analyses between manual and automatic data indicated Pearson correlation coefficients for biceps of 0.645-0.841 and 0.564-0.673 for lumbrical muscles. Relative fiber populations were accurate to a degree of +/- 4%. Conclusions: This protocol provides a reliable tool for quantification of muscle fiber populations. Using freely available software, it decreases the required time to analyze whole muscle sections.