Immunostaining for myofiber
Immunostaining for myofiber
Material
- Single myofiber in 48-well plate
See Preparation of single myofiber.
- 0.01% Triton/PBS (4C)
- 0.2% Triton/PBS (4C)
- 2% PFA/PBS (4C)
- 1% BSA/PBS (4C)
- 1/10,000 DAPI in 0.01% Triton/PBS (4C)
- Mounting medium (Dako S3023, 4C)
- Slide glass
- PAP pen
- Cover glass (24x50 mm)
Fixation
- Use 1 ml sol/well in following procedure.
- Fix by 2% PFA at RT for 15 min.
- Wash by 0.01% Triton 3 times.
- Permeabilize by 0.2% Triton at RT for 15 min.
- Wash by 0.01% Triton twice.
- Blocking by 1% BSA at RT for 30 min.
Primary antibody
- Prepare 200-500 ul primary antibody sol in 1% BSA at appropriate concentration.
See Antibody.
- Remove 1% BSA.
- Add primary antibody sol.
- Incubate at 4C for o/n.
Secondary antibody
- Prepare 200-500 ul secondary antibody sol in 1% BSA at appropriate concentration.
See Antibody.
- Wash by 0.01% Triton 3 times.
- Add secondary antibody sol.
- Incubate at RT for 1 h.
- Wash by 0.01% Triton twice.
- Add 20 ul DAPI (final 1/100,000).
- Incubate at RT for 5 min.
- Mask circumference of slide glass by PAP pen.
- Move myofiber onto slide glass.
- Remove extra sol by pipet.
- Add 1-2 drop of mounting medium.
- Enclose by cover glass.
