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EM Immunogold Labeling Protocol - P

时间:2006-01-22 15:55来源:ihcworld.com 作者:bioguider 阅读:
 

EM Immunogold Labeling Protocol - Post-embedding Method

 

Procedure:

 

1.    Fixation: fix specimen with 4% formaldehyde in 0.1M PB, pH 7.4 for 12-24 hours. For free cells or cell suspension, fix cell pellet for 2-4 hours.

2.     Rinse in 0.1M PB for 3x10 min.

3.     Quench Free Aldehyde: incubate specimen in 0.1M Glycine Solution for 20 minutes to quench the   free aldehyde groups. Note: this step may not be necessary.

4.    Incubate in 0.2M Sucrose Solution for 2x15min or overnight at 4C. Note: specimen can be stored in the sucrose solution for several weeks.

5.    Rinse in 0.1M PB briefly.

6.    Dehydration: incubate specimen in two changes of 70% ethanol 30 minutes each. Then incubate in L.R. White and 70% ethanol (2:1) mixture: slowly add one part of 70% ethanol drop by drop to two parts of L.R. White, and shake gently to avoid mixture becomes milky. Incubate specimen in the mixture for 1 hour.

7.    Transfer specimen in three changes of pure L.R. White for 1 hour each. Keep specimen in the last change of L.R. White overnight at room temperature (specimen can be stored in L.R. White at 4 C for weeks if necessary).

8.    L.R. White Embedding: place specimen in bottom of Gelatin Capsule, fill up with L.R. White to the brim, and slide the other half of the capsule on. Polymerize in 60 C oven for 24-48 hours or longer.

9.    Trimming: trim EM blocks and cut 0.5um thick sections and stain with toluidine blue if necessary, and then select the region of interest and trim blocks further to the appropriate size.

10.  Sectioning: cut ultrathin sections at 60-90 nm.

11.  Collect sections on formvar-carbon coated nickel grids (5 grids per specimen), and allow the grids to dry overnight prior to staining.

12.  Pretreatment: steam grids in Epitope Unmasking Solution (based on LM test or use citrate buffer pH 6.0) for 20 minutes, and allow grids to cool for 20 minutes. This step may not be necessary.

13.  Rinse sections for 2x2 min by place grids on large droplets of TBS-Tween.

14.  Serum Blocking: incubate sections in IHC Ultra Serum Block – Species match secondary antibody for 30 minutes.

15.  Primary Antibody: incubate sections on droplets of primary antibody diluted in Universal Antibody Diluent for 2 hours at room temperature. Note: primary antibody dilution is usually 10 times more concentrated than immunostaining at LM level.

16.  Rinse sections on large droplets of TBS-Tween for 6x2 min.

17.  Secondary Antibody: incubate sections on droplets of biotinylated secondary antibody (1:50, Vector Lab) in Universal Antibody Diluent for 1 hour at room temperature.

18.  Rinse on large droplets of TBS-Tween for 6x2 min.

19.  Streptavidin-Gold: incubate sections with gold conjugated streptavidin (1:20, EMS) in TBS, pH 7.6 for 1 hour. Note: do not use BSA or serum containing solution/reagent to dilute streptavidin-gold since BSA or serum may contain biotin, therefore reduce streptavidin-gold affinity to biotinylated secondary antibody.

20.  Rinse on large droplets of TBS-Tween for 6x2 min.

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