求教关于人胚胎干细胞免疫荧光的问题

ruofan1982

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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. 1 w, ]2 h- A0 E5 C! `
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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.
# N9 |& u5 k7 J  l2 J. N2. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. , ~5 s( d+ v1 z+ J& s5 A9 C
3. Permeabilize cells with 0.1% Triton X-100 / PBS for 10 minutes at room temperature. % C( t1 f; o* f
4. Wash twice (5-10 minutes each) with 1 X Rinse Buffer. : X  N- c  W8 j' f3 f* c" J% \
5. Apply Blocking Solution for 30 minutes at room temperature. 6 y) r. i+ v5 I# c
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. 8 D" j& x, h6 Q1 v( N) c' K) P
7. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
4 a! S" u  E7 E  @, C2 ]7 D8. Dilute secondary antibodies in 1 X PBS just before use. Incubate secondary antibodies for 30-60 minutes at room temperature. - \: r4 F3 Z: C
9. Wash three times (5-10 minutes each) with 1 X Rinse Buffer.
, l; e. W* a- q* I* ~( j; \$ E10. 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.
8 Z* I2 G; ^$ u, _) @; v$ W11. Fluorescence images can be visualized with a fluorescence microscope. 7 H9 m# M9 S5 n( g$ `$ h