Difference between revisions of "Antibody staining"
Jump to navigation
Jump to search
(Created page with "* 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 s...") |
|||
| Line 1: | Line 1: | ||
| − | + | == Procedure == | |
| − | + | # 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 | ||
Latest revision as of 10:39, 27 March 2015
Procedure
- 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