Antibody staining

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  1. Cut the tail and fix it in fresh 4% PFA in phosphate buffer from few hours to o/n always at 4C
  2. Wash in PBS 1 hour at room temperature, put tail in 30% sucrose Put in OCT store at –20C
  3. Cut cross sections of tail of 16μm-20μm
  4. Let dry at room temperature for 2 hours.
  5. Wash 3 times with TBS-tween (0.3% tween) for 5-10 min each
  6. Block the sections with TBS-Tween+ 20% goat serum for 1 hour at room temperature or o/n at 4C
  7. Thaw the antibody at room temperature or 37°C once thawed put on ice.
  8. Spin the tube at max speed for 10 minutes
  9. Add 100-200μL of antibody to each slide
  10. Leave for few hours at room temperature or o/n at 4 degree.
  11. Wash with TBS-tween 5 times for 10 min at room temperature
  12. Dilute the goat anti-mouse Cy5 1:200 in blocking buffer
  13. Apply 100-150 μL of antibody and leave 1 hour at room temperature
  14. Wash 5x with TBS-tween
  15. Add Hoechst 1:1000 in TBS tween for 3-5min, wash with TBS tween
  16. Mount