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非常感谢！！！
/ d8 s) Y$ h2 h1 M请问你封闭用的什么？buffer是怎么配的？+ F2 g6 k2 _" {* S8 d
我是按照抗体说明书操作的：
8 V* n- \9 K" _' w/ y5 IBuffer: Prepare a solution containing phosphate buffered saline, u: |2 o4 f5 Z, o6 g
(PBS) pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM3 K. A, e. R v; p! Z
EDTA by diluting MACS BSA Stock Solution (# 130-091-376)
! i* e6 J4 K' R1:20 with autoMACS™ Rinsing Solution (# 130-091-222). Keep
8 u) W; d7 c: _4 h# n) ?buffer cold (4−8 °C).8 Z$ P( x8 z( m n: V2 I9 N
▲ Note: EDTA can be replaced by other supplements such as anticoagulant' m$ z% I" g- @' O5 u- U a0 M
citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
! v: ]% h" ^! ^: OBSA can be replaced by other proteins such as human serum albumin, human: E+ o' M" z* e% r' u" ^
serum, or fetal calf serum. Buffers or media containing Ca2+ or Mg2+ are not) Q7 W& C' f; Z# d* @1 i
recommended for use.* ~5 G! }9 \* O Z( X# y: z7 ~- |
General protocol for immunofluorescent staining：" N" O1 B' ^8 o- ?6 C4 R7 N
1. Resuspend up to 10⁷ nucleated cells per 80 μL of buffer.5 S, v! A" @8 h4 }& w0 v& C
2. Add 20 μL of FcR Blocking Reagent.5 K0 R4 Q- w* N& H% S0 R5 Q9 v3 e4 E
3. Add 10 μL of the CD133/2 (293C3) antibody.
. D y# q* k& ]! P% M4. Mix well and refrigerate for 10 minutes in the dark (4−8 °C).
! f$ D i+ I% ~& b- b% ^9 k# ]▲ Note: Working on ice requires increased incubation times. Higher temperatures
4 _' |% `1 a( ~, Wand/or longer incubation times lead to non-specific cell labeling., m- W N% B# T9 j3 E
5. Wash cells by adding 1−2 mL of buffer per 10⁷ cells and
) a( r% V6 ]4 g& w4 m! r7 K' [: S$ ?2 ?8 vcentrifuge at 300×g for 10 minutes. Aspirate supernatant" k, D& e; w% Q& i+ S7 w
completely.3 ]7 H5 g S1 T* a6 X2 ?: ^3 L4 h |
6. (Optional) If CD133/2 (293C3)-Biotin was used, resuspend the
" A7 j2 Z+ C+ X: T. T/ l; U3 Jcell pellet in 100 μL of buffer, add 10 μL of anti-biotin antibody
; y1 {+ J6 h# g8 M* h- y2 u5 O) H(Anti-Biotin-FITC # 130-090-857, Anti-Biotin-PE # 130-090-
' I% B' `2 S* B756, or Anti-Biotin-APC # 130-090-856), and continue as( n$ ~ i% @, H
described in steps 4 and 5.: N1 t# e: P, `2 N( X; {, }
7. Resuspend cell pellet in a suitable amount of buffer for analysis( g$ f# ]5 j, r2 k d o8 F5 ?
by flow cytometry or fluorescence microscopy. |
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发表于 2011-9-23 23:57
