Table 1.
List of major antibodies and cocktail configurations used for MHC staining of rat, mouse, and human skeletal muscle.
Table 2.
Overview of immunofluorescence MHC staining protocol.
Figure 1.
Representative images of rat red tibialis anterior (RTA) muscle showing MHC expression as well as SDH and GPD activity staining.
Panel A, rat muscle serial cross-section incubated with a primary antibody cocktail against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. Shown are type I (blue), type IIA (green), type IIB (red), type IIX (unstained), and type IIAX (intermediate green) fibers. Panel B, rat muscle serial cross-section incubated with a primary antibody cocktail against MHCIIa (SC-71) and MHCIIx (6H1), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. This confirms the presence of type IIA (green) fibers, as well as confirms that the unstained fibers and intermediate green stained fibers in Panel A are type IIX (purple) and type IIAX fibers (green and purple), respectively. Panel C, rat muscle serial cross-section showing SDH activity staining. Panel D, rat muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.
Figure 2.
Representative images of mouse red gastrocnemius (RG) muscle showing MHC expression as well as SDH and GPD activity staining.
Panel A, mouse muscle serial cross-section incubated with a primary antibody cocktail against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. Shown are type I (blue), type IIA (green), type IIB (red), type IIX (unstained), and type IIXB (intermediate red) fibers. Panel B, mouse muscle serial cross-section incubated with a primary antibody cocktail against MHCIIa (SC-71) and MHCIIx (6H1), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. This confirms the presence of type IIA (green) fibers, as well as confirms that the unstained fibers and intermediate red stained fibers in Panel A are type IIX (purple) and type IIXB (purple and red) fibers, respectively. Panel C, mouse muscle serial cross-section showing SDH activity staining. Panel D, mouse muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.
Figure 3.
Representative images of rat, mouse, and human muscle showing type I/IIA hybrid fibers.
Panels A–C (rat RG), D–F (mouse soleus), and G–I (human VL) are images of muscle cross-sections incubated concurrently with primary antibodies against MHCI (BA-F8) and MHCIIa (SC-71) showing type I (blue), type IIA (green), and type I/IIA (blue and green) fibers. Bars represent 50 µm.
Table 3.
Quantitative analysis of fiber type distribution, CSA, SDH activity, and GPD activity in rat muscles.
Table 4.
Quantitative analysis of fiber type distribution, CSA, SDH activity, and GPD activity in mouse muscles.
Figure 4.
Bubble plot showing SDH activity, GPD activity, and CSA for each fiber type in mouse, rat, and human skeletal muscles.
Panel A, relative fiber type-specific SDH activity, GPD activity, and CSA for ten mouse skeletal muscles. Panel B, relative fiber type-specific SDH activity, GPD activity, and CSA for ten rat skeletal muscles. Panel C, relative fiber type-specific SDH activity, GPD activity, and CSA for human VL muscle. Data presented in this figure are from Tables 3, 4, and 5. Bubble size represents the relative CSA within a species. SDH and GPD activity are expressed relative to the values obtained in type I fibers (soleus for mouse and rat, VL for human) and assigned a reference value of 1.0.
Figure 5.
Representative images of human muscle showing MHC expression as well as SDH and GPD activity staining.
Panels A–D, merged and single channel images of a human vastus lateralis (VL) muscle serial cross-section incubated with an antibody cocktail (BA-F8, SC-71, and 6H1). Shown are type I (blue), type IIA (strong green), type IIX (strong red and intermediate green), and type IIAX (intermediate/strong green and intermediate red) fibers. *Note that the lower intensity staining for SC-71 in type IIX fibers is indicative of non-specific cross-reactivity (see results and discussion for further details). Panels E–H, merged and single channel images of a human VL muscle serial cross-section incubated with an antibody cocktail (BA-F8, BF-35, and 6H1). Shown are type I (blue), type I and type IIA (intermediate green and strong green, respectively), type IIX (strong red), and type IIAX (intermediate/strong green and intermediate red) fibers. Panel I, human VL muscle serial cross-section showing SDH activity staining. Panel J, human VL muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.
Table 5.
Quantitative analysis of fiber type distribution, CSA, SDH activity, and GPD activity in human vastus lateralis muscle.