2.4 Immunolabeling
of MuSCs
- 1PBS: 138 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ,
8mMNa 2 HPO 4 ·7H 2 O, pH 7.4. - Paraformaldehyde (PFA): 1PBS, 4% (w/v) PFA.
- Mounting medium.
- Permeabilization buffer: 1PBS, 0.5% (v/v) Triton X-100.
- Saturation buffer: 1PBS, 2% (w/v) bovine serum albumin
(BSA). - First antibody mix: 1PBS, 1% (w/v) BSA, 1/100 anti-Pax7
antibody (Hybridoma bank, concentrate), 1/50 anti-MyoD
antibody (Santa Cruz, #sc760) and 1/100 anti-Ki67 antibody
(Abcam, #ab15580) (seeNote 3)or60μg/ml anti-Desmin
antibody (Abcam #ab32362) or 1/50 anti-active form of cas-
pase 3 (Abcam, #ab32042). - Second antibody mix: 1PBS, 1/200 anti-Mouse IgG Biotin-
conjugated (Vector, #B1-2000), 1/200 anti-Rabbit IgG
Cy3-conjugated (Jackson, #711-165-152). - Streptavidin mix: 1 PBS, 0.1% (v/v) DTAF-conjugated
Streptavidin (Jackson, #016-010-084). - Nuclei staining solution: 1PBS, 2 nM (v/v) Hoechst (see
Note 4).
3 Methods
3.1 MuSCs
Extraction
From one adult healthy mice (3–6-month old) using the two hind
limbs, 50,000–120,000 MuSCs can be sorted.- Keep on ice a 30 ml container and petri dishes. Fill the petri
dishes with 5 ml of ice-cold 1PBS (seeNote 5). - Prepare digestion buffer and preheat at 37C.
- Euthanize mice according to your local regulation. Harvest
muscles of both hind limbs (includingtibialis anterior,gastroc-
nemius,quadriceps,ischio, andgluteus), and put them in the
petri dish previously filled with 1PBS. Then discard fat,
tendons, fascia, vessels, and nerves (seeNote 6). - Pour muscle (without taking the PBS) in the cold 30 ml con-
tainer, and crush in very small pieces to obtain a pulp. - Add 8 ml of the digestion buffer on the muscle slop and
incubate for up to three rounds of 10 min in a shaking water
bath at 37C(seeNote 7). - Vortex the mixture between each round.
- From here, all the next steps have to be made on ice and in
sterile conditions. - Stop the digestion by adding 2 ml of FBS. Mix by pipetting up
and down.
542 Marine Theret et al.