Livesey: Antigen retrival: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
mNo edit summary
(wiki markup)
 
Line 1: Line 1:
Adapted from Hevner Protocol
Adapted from Hevner Protocol
 
# Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
# Leave the chosen slides at room temperature just until the frost melts.
 
# Hydrate slides in 1x PBS for 10 seconds.
2. Leave the chosen slides at room temperature just until the frost melts.
# Put slides into '''0.01 M Citric acid (pH 6.0), microwave''' just to a boil, and let cool for 5 minutes.
 
# Repeat Step 4 two more times (three times total).
3. Hydrate slides in 1x PBS for 10 seconds.
# Rinse the slides 6 times in 1x TBST for 8 minutes each.  
 
# Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
4. Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.
# Incubate overnight at 4ºC with primary antibodies in TBST plus 5% milk.
 
# Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
5. Repeat Step 4 two more times (three times total).
# Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
 
# Rinse 3 times in 1x TBS for 8 minutes each.
6. Rinse the slides 6 times in 1x TBST for 8 minutes each.  
# Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
 
# Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
 
8. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
 
9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
 
10. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
 
11. Rinse 3 times in 1x TBS for 8 minutes each.
 
12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
 
13. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
 
 
TBST = TBS + 0.1% Triton
TBST = TBS + 0.1% Triton

Latest revision as of 08:34, 19 October 2009

Adapted from Hevner Protocol

  1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
  2. Leave the chosen slides at room temperature just until the frost melts.
  3. Hydrate slides in 1x PBS for 10 seconds.
  4. Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.
  5. Repeat Step 4 two more times (three times total).
  6. Rinse the slides 6 times in 1x TBST for 8 minutes each.
  7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
  8. Incubate overnight at 4ºC with primary antibodies in TBST plus 5% milk.
  9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
  10. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
  11. Rinse 3 times in 1x TBS for 8 minutes each.
  12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
  13. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.

TBST = TBS + 0.1% Triton

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Livesey:_Antigen_retrival&oldid=360246"