|
- 积分
- 14
- 威望
- 14
- 包包
- 209
|
非常感谢！！！
% l& z j: t6 M! N! @) S请问你封闭用的什么？buffer是怎么配的？" c3 W, C/ j( |
我是按照抗体说明书操作的：7 N, F# o( {% B& [; U8 _6 f+ K
Buffer: Prepare a solution containing phosphate buffered saline: E) i1 I# u5 U' Z0 y
(PBS) pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM
}+ b; y: x7 hEDTA by diluting MACS BSA Stock Solution (# 130-091-376)6 L2 n; X4 }" k- B/ t8 |
1:20 with autoMACS™ Rinsing Solution (# 130-091-222). Keep8 H" s5 r) w U
buffer cold (4−8 °C).' p$ a! U2 T+ C7 p7 F
▲ Note: EDTA can be replaced by other supplements such as anticoagulant$ ?6 M6 I: D1 \$ Y2 ?* l
citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).; X4 \$ V3 i2 z% r
BSA can be replaced by other proteins such as human serum albumin, human& F% _. L/ I7 ~' I) C% F9 P
serum, or fetal calf serum. Buffers or media containing Ca2+ or Mg2+ are not$ }1 x* j' P' |) {
recommended for use.
' f* A8 H$ M. }. z% O' n+ K General protocol for immunofluorescent staining：- g. X; `# s# G: l- I, T4 E, a4 H
1. Resuspend up to 10⁷ nucleated cells per 80 μL of buffer.
4 O" I! Z' S; c2. Add 20 μL of FcR Blocking Reagent.
! G5 P) F5 L) k% ~# a3. Add 10 μL of the CD133/2 (293C3) antibody.
+ Y7 h. X v% S7 ]9 y+ l% D4. Mix well and refrigerate for 10 minutes in the dark (4−8 °C).
, J3 V% G1 D+ Y▲ Note: Working on ice requires increased incubation times. Higher temperatures
& r2 K2 }! C+ aand/or longer incubation times lead to non-specific cell labeling.& j8 B& j, S8 m4 v5 Q
5. Wash cells by adding 1−2 mL of buffer per 10⁷ cells and) p' @7 P% ` t0 l9 i/ p2 R# u
centrifuge at 300×g for 10 minutes. Aspirate supernatant
4 l! W& o: Z @: o/ ]completely./ T; U! a$ m- ?/ z. K: Q
6. (Optional) If CD133/2 (293C3)-Biotin was used, resuspend the
8 \9 r8 z/ T( jcell pellet in 100 μL of buffer, add 10 μL of anti-biotin antibody K& K3 P# q7 K" N- M5 O2 |
(Anti-Biotin-FITC # 130-090-857, Anti-Biotin-PE # 130-090-$ P, x6 h4 u/ e
756, or Anti-Biotin-APC # 130-090-856), and continue as2 h& }- X$ I2 l4 D" C
described in steps 4 and 5.* c4 ?% W/ H% F" k- A& N
7. Resuspend cell pellet in a suitable amount of buffer for analysis
% `- Y( B7 c/ m; o3 Z- h4 ?by flow cytometry or fluorescence microscopy. |
-
总评分: 威望 + 2
包包 + 10
查看全部评分
|
发表于 2011-9-23 23:57
