EAAT1/GLAST-1/SLC1A3 Antibody - BSA Free

Culture cells to appropriate density on suitable glass coverslips in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and gently add 10% formalin to cover the cells. Fix at room temperature for 5-10 minutes.
2. Remove the formalin and add 0.5% Triton-X 100 in TBS to permeabilize the cells. Incubate for 5-10 minutes.
3. Remove the permeabilization buffer and add wash buffer (PBS or PBS with 0.1% Tween-20). Be sure to not let the cells dry out. Gently wash three times for 10 minutes.
4. Alternatively, cells can be fixed with -20 degrees C methanol for 10 minutes. Remove the methanol and rehydrate in PBS for 10 minutes before proceeding.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum for 60 minutes at room temperature.
6. Add primary antibody at the appropriate dilution and incubate at room temperature for 60 minutes or at 4 degrees C overnight.
7. Remove primary antibody and replace with wash buffer. Gently wash three times for 10 minutes.
8. Add secondary antibody at the appropriate dilution. Incubate for 60 minutes at room temperature.
9. Remove antibody and replace with wash buffer. Gently wash three times for 10 minutes.
10. Nuclei (DNA) can be stained with 4',6' diamino phenylindole (DAPI) at 0.1 ug/ml, or coverslips can be directly mounted in media containing DAPI.
11. Cells can now be viewed with a fluorescence microscope.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow proper laboratory procedures for the disposal of formalin.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 60 minutes at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 degrees C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.

Western Blot Protocol
1. Perform SDS-PAGE on protein samples to be analyzed, loading 10-40 ug of total protein per lane.
2. Electro-blot the proteins to a suitable membrane (PVDF or Nitrocellulose) according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or a similar product) to assess transfer success. Mark molecular weight standards where appropriate.
4. Thoroughly rinse the membrane of stain with TBST.
5. Incubate the membrane in blocking buffer (5% non-fat milk in TBST or 5% BSA in TBST) as appropriate, for 60 minutes.
6. Dilute the SLC1A3 primary antibody as appropriate in blocking buffer and incubate for 60 minute at room temperature to overnight at 4 degrees C with gently shaking.
7. Wash the membrane in TBST three times for 10 minutes each.
8. Incubate the membrane in the appropriate secondary antibody prepared in blocking buffer (as per manufacturer's instructions) and incubate for 60 minutes at room temperature.
9. Wash the membrane in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
10. Incubate the membrane in the appropriate detection reagent in accordance with the manufacturer's instructions and image the blot.
Note: Tween-20 can be added to the blocking, wash and antibody dilution buffers to a final concentration of 0.05-0.1%.