Table 1.

Antibodies, concentrations, and immunohistochemistry protocols used to label muscle fibers

Primary antibodies and concentrations	Reactivity	Secondary antibodies and concentrations
BF-F8 (1:100)*	I	Alexa488 IgG2b, goat/anti-mouse (1:200)
BF-F3 (1:100)*	IIb	Alexa Fluor 594, IgG1, goat/anti-mouse (1:200)
6H1 (1:50)*	IIx	Alexa Fluor 594, IgG1, goat/anti-mouse (1:200)
SC-71 (1:100)*	IIa	Alexa488 IgG1, goat/anti-mouse (1:200)
MHC13 (1:50)***	IIeo	Alexa488 IgG1, goat/anti-rabbit (1:200)
anti-laminin (1:200)**	laminin	Alexa405, goat/anti-rabbit IgG Invitrogen (1:100)
Targets	Procedure	Time
1. MHC I and IIb and laminin	Phosphate buffer (PB) wash	2 × 2 min
	Cold (4°C) methanol fixation	10 min
	PB wash	2 × 2 min
	Incubate in 10% normal goat serum in PB	1 hr
	PB wash	2 × 2 min
	Incubate in primary antibodies at 4°C	overnight
	PB wash	4 × 2 min
	Incubate in secondary antibodies at RT	30 min
	PB wash	3 × 2 min
	Mount coverslips with Prolong Gold antifade reagent
1. MHC IIa and IIx and laminin	Air dry	10 min
2. MHC IIeo	Incubate in 10% normal goat serum in phosphate buffered saline (PBS)	1 hr
	Incubate in primary antibodies at RT	2 hrs
	PBS wash	3 × 5 min
	Incubate in secondary antibodies at RT	1 hr
	PBS wash	3 × 5 min
	Mount coverslips with ProlongH Gold antifade reagent

Primary and secondary antibody cocktails were diluted in blocking buffer

^*Antibodies obtained from (Developmental Studies Hybridoma Bank, Iowa City, IA)

^**Antibodies obtained from (Sigma-Aldrich, St. Louis, MO)

^***Anitbodies obrain from (KY)

^****Antibodies obtained from (Invitrogen: Molecular Probes, Eugene, OR)