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. M: n$ s" Z8 j X8 T5 {, LThe 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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Fluorescein 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. 8 g$ X) i1 i B) L' L7 ]
2. Wash twice (5-10 minutes each) with 1 X Rinse Buffer.
3 S$ {: @ u0 z; K3. Permeabilize cells with 0.1% Triton X-100 / PBS for 10 minutes at room temperature.
5 s3 m# b( `# w5 b+ A0 V4. Wash twice (5-10 minutes each) with 1 X Rinse Buffer.
& T9 P$ I9 t/ [7 i5. Apply Blocking Solution for 30 minutes at room temperature.
; D7 T8 C; K0 E5 E' P6. 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. ! y/ Z* J5 o( |5 Z* ^) k
7. Wash three times (5-10 minutes each) with 1 X Rinse Buffer. 8 o, W- k) {. U( l* Y& W
8. Dilute secondary antibodies in 1 X PBS just before use. Incubate secondary antibodies for 30-60 minutes at room temperature. 2 Q7 ~) E, }+ A& X
9. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
' x# }7 m; I7 c) S& _& F- Y+ ~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. : j! U7 o D/ j7 b; C! U* h
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
