From: MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool
Primary antibody | Secondary antibody | ||
---|---|---|---|
Fiber/nuclei (from 2 labs) | Laminin | Laminin α-2 (4H8-2) Santa Cruz #59854 (1/250) ON 4 °C | Donkey anti-rat IgG Alexa Fluor 488 ThermoFisher #A-21208 (1/500) 45 min RT |
Hoechst | Hoechst 33342 ThermoFisher #H3570 (1/1500) 45 min RT | ||
Laminin | Anti-laminin Ab (produced in Rabbit) L9393 thermoscientific (1/100) ON 4 °C | Pierce donkey anti-rabbit IgG (H+L) cross adsorbed secondary Ab DyLight 650 (prod#Sa5-1041) (1/200) 1 h RT | |
Hoechst | Hoechst 33342 ThermoFisher #H3570 (1/1000) 1 h RT | ||
Satellite cell | Laminin | Anti-laminin Ab (produced in rabbit) L9393 thermoscientific (1/100) ON 4 °C | Pierce donkey anti-rabbit IgG (H+L) cross adsorbed secondary Ab DyLight 650 (prod#Sa5-1041) (1/200) 1 h RT |
Pax7 | Monoclonal mouse anti-Pax7(supernatant), Developmental Studies Hybridoma Bank (1/20) ON 4 °C | Biotinylated horse anti-mouse IgG (H + L) (Vector Laboratories, #BA-2000) = amplification (1/200) 1 h RT + Streptavidin-DTAF (Beckman Coulter, #PN IM0307) (1/1000) 1 h RT | |
Vessels | Laminin | Anti-laminin Ab (produced in rabbit) L9393 thermoscientific (1/100) ON 4 °C | Pierce donkey anti-rabbit IgG (H + L) cross adsorbed secondary Ab DyLight 488 (prod#Sa5-10038) |
CD31 | BD Pharmigen 550,274 purified rat anti-mouse CD31 (1/50) ON 4 °C | Pierce donkey anti-rat IgG (H + L) cross adsorbed secondary Ab DyLight 550 (Prod#Sa5-10027) (1/200) 1 h RT | |
Fiber type | Type I | BA-D5 (mouse IgG2b) DSHB (1/100) 1 h 37 °C | Anti-mouse IgG2b 647 nm life technology A21242 (1/250) 30 min 37 °C |
Type IIA | SC-71 (mouse IgG1) DSHB (1/100) 1 h 37 °C | Anti-mouse IgG1 568 nm life technology A21124 (1/250) 30 min 37 °C | |
Type IIB | BF-F3 (mouse IgM) DSHB (1/100) 1 h 37 °C | Anti-mouse IgM 488 nm life technology A21042 (1/250) 30 min 37 °C | |
Laminin | anti-laminin (rabbit) DSHB(1/100) 1 h 37 °C | Anti-rabbit 4+A2:D1905nm abcam ab175651 (1/250) 30 min 37 °C |