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The cell-surface-antigen expression of cultured cells can be analyzed by using immunofluorescence techniques. The following primary monoclonal antibodies are used to detect surface-antigen expression: anti-SSEA-1;anti-SSEA-3; anti-SSEA-4; anti-TRA-1-60; anti-TRA-1-81; and anti-Oct-4. 7 \, u7 N; B3 ]- u! q
) ?7 W x9 p, fFluorescein isothiocyanate (FITC)-labeled secondary antibodies, appropriate to the species and isotype of the primary antibody, can be used for detection of primary antibody binding. ! q$ [( ?/ v, u+ M# X& q [1 c
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1. Fix cultured ES cells in Fixative Solution for 15-20 minutes at room temperature. , e8 d8 j: p) m" P9 ^: Z" ~
2. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. * w: L/ a9 R) w: F
3. Permeabilize cells with 0.1% Triton X-100 / PBS for 10 minutes at room temperature.
8 Z3 B9 S, z9 j, n4. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. 2 P% Q+ y+ a6 c
5. Apply Blocking Solution for 30 minutes at room temperature.
7 v. ^# D& E, {) D6. Dilute primary antibodies to working concentrations in Blocking Solution (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 & Oct-4 can be used in the range of 1:10 – 1:50). Incubate primary antibodies for 1 hour at room temperature. ; G3 \5 K5 x. T; C
7. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
5 N# ?4 y' q, H, Y9 l2 @- d8. Dilute secondary antibodies in 1 X PBS just before use. Incubate secondary antibodies for 30-60 minutes at room temperature.
4 c$ a( Z, a/ Y( w- D3 Y& f/ _9. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
* P! I4 S: ~, [) |10. If stained in plate wells, cells should be covered with 1 X PBS for visualization. However, if cells are stained on a coverslip, mount on a slide by using antifade mounting solution.
& N, Y, Z+ `( P11. Fluorescence images can be visualized with a fluorescence microscope. ) P ~; }% `9 \$ `. y' N
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发表于 2012-7-8 15:11
发表于 2012-8-19 20:51
