Immunostaining of integrin in 3T3 on glass coverslip Keiko Ookawa-Chinzei
Rev. 1, Apr 4, 2002
(for 12-well plate, 18 mm circle glass coverslips)
1. Aspirate medium, and wash in PBS.
2. Fix with 4% paraformaldehyde in PBS[i] at room temp for 30 min.
3. Permealize cells with 0.1% Triton X-100 in PBS for 5 min at room temp.
4. Rinse with PBS (10 min x 3 times).
5. Incubate samples in blocking buffer (1% normal goat serum, 0.1% Tween 20 in PBS) for 60 min at room temp to decrease the nonspecific adsorption of antibodies. If you prefer 2-day protocol, just incubate them overnight at 4 C but securely seal the well plate with ParaFilm.
6. Incubate samples with primary antibody (at optimal dilution in blocking buffer[ii]) for 60 min at room temp. For 18 mm coverslip in 12-well dish, add 100 uL diluted antibody per well and put coverslip “face down”. Seal the well plate with ParaFilm.
7. Wash samples with blocking buffer (10 min x 3 times). First add 2 mL of buffer, and then carefully put coverslips “face up” with 21G needle, and wait for 10 min, aspirate, add buffer….so on.
8. Incubate samples with secondary antibody (at optimal dilution in blocking buffer[iii]) for 30 min at room temp. As in 6, add 100 uL of diluted antibody per well and put coverslip “face down” and seal with ParaFilm. Cover with Al-foil to avoid fading.
9. Wash samples with PBS (10 min x 3 times). To preserve samples, keep them in 0.1% Na-azide in PBS and refridgerate.
[i] To prepare 4% PFA, add 4 g of paraformaldehyde to 90 mL PBS. Stir and heat to 60 C in a hume hood. Add NaOH pellet (or 10 M solution) and mix until the liquid is clear. After cooling, adjust pH to 7.0 and fill up to 100 mL. Use within 1 week.
[ii] For Pharmingen 550531 (integrin beta1 chain), 1:100.
[iii] For Jackson ImmunoResearch 712-075-150 (TexasRed anti-rat IgG) 1:50-100.