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X! v8 M! f3 v! F9 o2 t" ZThe 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 ~! {% {$ U4 O+ u) uFluorescein 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.
p) H" F, d5 | e1 D( t }2. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. ; y/ n: R/ r8 Y) \. G0 ~4 i
3. Permeabilize cells with 0.1% Triton X-100 / PBS for 10 minutes at room temperature. # D3 Z$ L' W6 K* h7 V
4. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. 3 m( R+ g$ W( F4 r* H- o) X9 l& f
5. Apply Blocking Solution for 30 minutes at room temperature. + i; o3 Z7 x% z4 |$ H' B5 b% y
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. 0 _; x* y d" s& ]5 U: C# r
7. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
2 d4 M) o2 k0 I& H! t' m8. Dilute secondary antibodies in 1 X PBS just before use. Incubate secondary antibodies for 30-60 minutes at room temperature.
9 ^& ~: f: c$ K1 K# N4 i i& G9. Wash three times (5-10 minutes each) with 1 X Rinse Buffer. $ T) |- }; B! Q! K* @6 F; I
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. / f& o& d& c7 w" o6 @/ n2 v
11. Fluorescence images can be visualized with a fluorescence microscope. ( {0 m- c8 i1 c# E a# {) ]2 d
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发表于 2012-7-8 15:11
发表于 2012-8-19 20:51
