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. Muscle Nerve 54: 292-299, 2016.
['Bergmeister|Konstantin D|KD|0000-0003-3910-9727', 'Gröger|Marion|M|', 'Aman|Martin|M|', 'Willensdorfer|Anna|A|', 'Manzano-Szalai|Krisztina|K|', 'Salminger|Stefan|S|', 'Aszmann|Oskar C|OC|']
D000818:Animals D003936:Diagnosis, Computer-Assisted D003952:Diagnostic Imaging D007150:Immunohistochemistry D008297:Male D018485:Muscle Fibers, Skeletal D018995:Myosin Heavy Chains D051381:Rats D017207:Rats, Sprague-Dawley D012044:Regression Analysis
muscle fiber type ImageJ rat myosin heavy chain fiber population automated muscle fiber analysis