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Post-Imbedding Immunoelectron Microscopy I use a sheet of parafilm pressed onto a 96 well plate to make indentations for the drops of solutions on
 

Summary: 110
Post-Imbedding Immunoelectron Microscopy
I use a sheet of parafilm pressed onto a 96 well plate to make indentations for the drops of solutions on
which the grids are floated. Place the 96 well dish in a plastic box with wet filter paper on the bottom
to humidify the interior. I use drops of 50 l volume. A self-closing, non-capillary forceps is best. I
don't dry the grid between the different steps. All buffers below are filtered through a 0.45 m filter.
Washing may be done with a gentle stream or "jet" instead of transfers between drops.
TBS may work better that PBS with some antibodies.
1. (Optional) Etch: 10 - 60 min at room temperature (RT) with freshly prepared 10% NaIO4. (I
usually use 15 minutes.)
2. Wash 3 X 2 minutes with water.
3. Block 15 - 30 minutes with PBS + 1% BSA + 0.01% TX-100 + NaN3.
4. Incubate with antibody for 1 - 2 hours at RT. Dilute antibody 1:2 to 1:50 in PBS + 1% BSA +
0.01% TX-100 + NaN3. Use monoclonal supernatant without dilution.
5. Wash 1 X 1 minutes + 4 X 5 minutes with PBS + 0.1% BSA + 0.01% TX-100 + NaN3. (The wash
buffer has a lower BSA concentration.)
6. Incubate with colloidal gold secondary antibody for 1 hr at RT. Dilute antibody 1:10 to 1:25 in PBS
+ 1% BSA + 0.01% TX-100 + NaN3.
7. Wash 1 X 1 minutes + 4 X 5 minutes with PBS + 0.1% BSA + 0.01% TX-100 + NaN3.
8. Wash 4 X 2 minutes in PBS.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine