Difference between revisions of "Simple"

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2. Leave the chosen slides at room temperature just until the frost melts.
 
2. Leave the chosen slides at room temperature just until the frost melts.
  
6. Rinse the slides 3 times in 1x TBST for 8 minutes each.  
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3. Rinse the slides 3 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.
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4. 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.
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5. 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.
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6. 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.
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7. 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.
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8. Rinse 3 times in 1x TBS for 8 minutes each.
  
12. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
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9. 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.
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10. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.

Latest revision as of 04:08, 27 June 2006

1. Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).

2. Leave the chosen slides at room temperature just until the frost melts.

3. Rinse the slides 3 times in 1x TBST for 8 minutes each.

4. Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.

5. Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.

6. Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.

7. Add secondary antibodies in TBST for 1 hour at room temperature in the dark.

8. Rinse 3 times in 1x TBS for 8 minutes each.

9. Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.

10. Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.