(1)Primary Antibody Adding
1. Discard all medium in the wells.
2. Washed the cells gently with PBS. For wells with nanoparticles, repeat this step 2 times. For wells without nanoparticles, do this step just once. Discard all PBS.
3. Fix the cells using 3% paraformaldehyde for 30 minutes at room temperature.
4. Washed the cells gently with PBS 3 times.
5. Add primary antibody containing solution(350μl/well).
6. Incubate the cells for 2 hours at room temperature.
Primary antibody containing solution(concentration: 1:1000):
a). NR2B antibody
b). PBS with 0.1% Triton X
c). 2% normal goat serum (NGS)
1ml primary antibody containing solution
= 1μl NR2B antibody + 20ul NGS + 979ul 0.1% TritonX PBS
350μl primary antibody containing solution/well X 26 wells =9100μl primary antibody containing solution
Primary antibody containing solution preparation:
10ml primary antibody containing solution
= 10μl NR2B antibody + 200ul NGS + 9790ul 0.1% TritonX PBS
(2)Secondary Antibody Adding
7. Discard all primary antibody containing solution.
8. Wash with PBS three times.
9. Add secondary antibody containing solution(350μl/well).
10. Incubate the cells for 2 hours at room temperature.
Fluorescein(Alexa 488)-conjugated goat anti-rabbit secondary antibody
Secondary antibody containing solution(concentration: 1:500):
a). Fluorescein-conjugated goat anti-rabbit secondary antibody
1ml secondary antibody concentration
= 2μl of goat anti rabbit Alexa 488 + 998μl PBS (bench use PBS)
350μl secondary antibody containing solution/well X 26 wells = 9100μl secondary antibody containing solution
Secondary antibody containing solution preparation:
10ml secondary antibody containing solution
=20μl of goat anti rabbit Alexa 488 + 9980μl PBS (bench use PBS)
11. Discard all secondary antibody containing solution and wash with PBS three times.
12. Add one drop mounting medium (DAKO) onto a clean slide.
13. Coverslips with immunostained cells were mounted on the clean slide (without bubbles).