Livesey: Antigen retrival

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Current revision (10:34, 19 October 2009) (view source)
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1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
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Adapted from Hevner Protocol
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# Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
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2. Leave the chosen slides at room temperature just until the frost melts.
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# Leave the chosen slides at room temperature just until the frost melts.
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# Hydrate slides in 1x PBS for 10 seconds.
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3. Hydrate slides in 1x PBS for 10 seconds.
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# Put slides into '''0.01 M Citric acid (pH 6.0), microwave''' just to a boil, and let cool for 5 minutes.
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# Repeat Step 4 two more times (three times total).
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4. Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.
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# Rinse the slides 6 times in 1x TBST for 8 minutes each.  
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# Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
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5. Repeat Step 4 two more times (three times total).
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# Incubate overnight at 4ºC with primary antibodies in TBST plus 5% milk.
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# Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
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6. Rinse the slides 6 times in 1x TBST for 8 minutes each.  
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# Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
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# Rinse 3 times in 1x TBS for 8 minutes each.
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7. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
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# Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
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# Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
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8. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
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9. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
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10. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
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11. Rinse 3 times in 1x TBS for 8 minutes each.
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12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
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13. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
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TBST = TBS + 0.1% Triton
TBST = TBS + 0.1% Triton

Current revision

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

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