Antibody staining

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