分子細胞機能学研究室

ホーム研究実験業績メンバーリンク

Immunostaining for section

Material

• 0.01% Triton/PBS (4C)
• 0.2% Triton/PBS (4C)
• 2% PFA/PBS (4C)
• Acetone (-20C)
• 1% BSA/PBS (4C)
• 10% BSA/PBS (4C)
• 1/10,000 DAPI in 0.01% Triton/PBS (4C)
• MOM
• Mounting medium (Dako S3023, 4C)
• Slide glass
• PAP pen
• Cover glass (24x50 mm)

Fixation

1. Fix by 2% PFA at RT for 5 min, OR
   Fix by acetone on ice for 10 min.
2. Wash by 0.01% Triton twice.
3. FOR MOUSE PRIMARY ANTIBODY: MOM at RT for 1 h.
4. Wash by 0.01% Triton twice.
5. Blocking by 5% serum in 10% BSA at RT for 30 min.

Primary antibody

1. Prepare 200 ul primary antibody sol/slide in 1% BSA at appropriate concentration.
   See Antibody.
2. Remove blocking sol.
3. Add primary antibody sol.
4. Incubate at 4C for o/n.

Secondary antibody

1. Prepare 200 ul secondary antibody sol/slide in 1% BSA at appropriate concentration.
   See Antibody.
2. Wash by 0.01% Triton twice.
3. Add secondary antibody sol.
4. Incubate at RT for 1 h.
5. Wash by 0.01% Triton twice.
6. Add 200 ul DAPI.
7. Incubate at RT for 5 min.
8. Remove DAPI.
9. Mask circumference of slide glass by PAP pen.
10. Add 1-2 drop of mounting medium.
11. Enclose by cover glass.