Perfusion staining methods for visualization of the intact microvascular networks in whole mount skeletal muscle preparations.

Introduction: Visualization of the intact microvascular network in skeletal muscle requires labeling the entire network in whole mount preparations where muscle fibre length can be set to near optimal but the tools to do this are not clear.

Methods: We intravascularly injected CD-1 mice with different fluorescently labelled lectins (fluorescent isolectin GS-IB4 (ISO), wheat germ agglutinin (WGA), lycopersicon esculentum (tomato) (LYCO) or FITC-labelled gel. Soleus, extensor digitorum longus, diaphragm, gluteus maximus and cremaster muscles were excised, pinned at optimal sarcomere length and viewed using fluorescence microscopy.

Results: WGA and LYCO were the effective at labeling the entire vascular network with WGA labeling capillaries more bright. ISO labelled the arteriolar vasculature and early segments of the capillaries but not the full length of the capillaries or the venular network. FITC-labelled gel was effective at labelling the microvascular network but not all small vessels were consistently labelled. The pattern of staining for each labelling method was similar across all muscle fibre-types tested.

Conclusions: WGA was optimal for perfusion labeling and visualization of the intact microvascular network in whole mount skeletal muscle preparations and can be used in combination with ISO to distinguish the arteriolar and venous sides of the network.