Antigen retrieval: Difference between revisions
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== Protocol == | |||
# 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. | |||
# 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). | |||
# 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. | |||
# 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. | |||
# Add secondary antibodies in TBST for 1 hour at room temperature in the dark. | |||
# Rinse 3 times in 1x TBS 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. | |||
TBST = TBS + 0.1% Triton | TBST = TBS + 0.1% Triton |
Revision as of 06:32, 19 October 2009
Protocol
- 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.
- 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).
- 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.
- 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.
- Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
- Rinse 3 times in 1x TBS 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.
TBST = TBS + 0.1% Triton