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EM Immunogold Labeling Protocol - P

时间:2006-01-22 15:57来源:ihcworld.com 作者:bioguider 阅读:
EM Immunogold Labeling Protocol - Pre-embedding Method

 

Procedure:

 

1.       Sectioning: Vibratome sections at 50 um thick.

2.       Collect sections in 0.1M PB.

3.       Pretreatment: treat sections with 1% sodium borohydride solution for 30 minutes.

4.       Rinse many times in 0.1M PB to remove bubbles.

5.       Rinse in PBS for 3x5min

6.       Serum Blocking: incubate in blocking buffer for 30 minutes.

7.       Primary Antibody: incubate sections with primary antibody (appropriate diluted in blocking buffer) overnight at room temperature.

8.       Rinse in PBS for 3x5min.

9.       Washing Buffer: incubate in washing buffer for 30 minutes.

10.    Secondary Antibody: incubate sections in gold-conjugated secondary antibody diluted 1:50 in washing buffer for 3 hours.

11.    Rinse 4x5 min in washing buffer and then 4x5 min in PBS.

12.    Post-fixation1: fix sections with 2% glutaraldehyde in PBS for 10 minutes.

13.    Rinse 4x5 minutes in PBS.

14.    Silver enhancement:

·          Rinse in 0.2M citrate buffer 3x2min.

·          Incubate in silver enhancement solution for 3-9 minutes for EM and 6-12 minutes for LM (If room temperature is very low, a longer time may be needed such as 20-30 min).

·          Rinse in 0.2M citrate buffer 2x5min

·          Rinse in 0.1 M PB 4x5 min

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