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: j, x J( @3 QThe 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.
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2 b" ]) ~- N3 wFluorescein isothiocyanate (FITC)-labeled secondary antibodies, appropriate to the species and isotype of the primary antibody, can be used for detection of primary antibody binding.
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1. Fix cultured ES cells in Fixative Solution for 15-20 minutes at room temperature.
! g6 z. v, ?6 _3 O E2. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. ' A: g" m1 i. W
3. Permeabilize cells with 0.1% Triton X-100 / PBS for 10 minutes at room temperature. " h0 Z" E; c- [3 T* ~8 K
4. Wash twice (5-10 minutes each) with 1 X Rinse Buffer.
* N: c o6 K) t( X2 P5. Apply Blocking Solution for 30 minutes at room temperature. 2 V6 i: m* u5 M5 Y6 @
6. 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.
9 t6 h: g' I, S7. Wash three times (5-10 minutes each) with 1 X Rinse Buffer. 4 g4 W) [) Y. I: o9 J* i
8. Dilute secondary antibodies in 1 X PBS just before use. Incubate secondary antibodies for 30-60 minutes at room temperature.
& B: q# b5 q! }! l9. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
A: H7 M) H- w, Z* k( V10. 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. }+ f, y$ }2 |! y7 s: n
11. Fluorescence images can be visualized with a fluorescence microscope.
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发表于 2012-7-8 15:11
发表于 2012-8-19 20:51
