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非常感谢！！！$ ]% V% D8 G9 ^+ C x- @
请问你封闭用的什么？buffer是怎么配的？7 J( F8 H8 G" t+ p! Z4 |
我是按照抗体说明书操作的：
. _5 A2 @1 d5 z0 o* d. Z" jBuffer: Prepare a solution containing phosphate buffered saline
1 @3 ]6 Y* H& b9 T1 W(PBS) pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM& L3 K8 s. u6 {' s1 u& L: V1 n1 n! Q
EDTA by diluting MACS BSA Stock Solution (# 130-091-376)( P% U2 E0 t* n) {) h
1:20 with autoMACS™ Rinsing Solution (# 130-091-222). Keep
9 N! O) l/ \/ wbuffer cold (4−8 °C).
- A. P3 X5 N: v% y. F* Y▲ Note: EDTA can be replaced by other supplements such as anticoagulant, Z; t$ K; U. A) U, D* D Q
citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD)." ~4 v8 z6 _8 K$ J8 f9 K' j& L+ s, L
BSA can be replaced by other proteins such as human serum albumin, human, f( H; h, x) D0 ]
serum, or fetal calf serum. Buffers or media containing Ca2+ or Mg2+ are not
/ _2 ^4 q. Q* Q* X0 drecommended for use. `& z! o; D: K& |7 B) g
General protocol for immunofluorescent staining：
7 H6 W: C4 \+ G% }1. Resuspend up to 10⁷ nucleated cells per 80 μL of buffer.
- N U" E3 z2 W/ ^. m" `! j2. Add 20 μL of FcR Blocking Reagent.
, t, i1 i8 K( t+ [3. Add 10 μL of the CD133/2 (293C3) antibody.
7 d6 \) N7 S1 k6 X" a' d' i1 ? C4. Mix well and refrigerate for 10 minutes in the dark (4−8 °C).
+ h4 V: d5 |' R2 U' o▲ Note: Working on ice requires increased incubation times. Higher temperatures/ z% V" T+ P- k4 |
and/or longer incubation times lead to non-specific cell labeling.
) r5 F2 p2 X& W5. Wash cells by adding 1−2 mL of buffer per 10⁷ cells and+ u; b6 P: e1 h+ E( r4 X- E( f
centrifuge at 300×g for 10 minutes. Aspirate supernatant
9 d$ G8 a% }( {+ _0 scompletely.
$ x0 S3 H# F) @3 }' U9 I5 M! g2 ?6. (Optional) If CD133/2 (293C3)-Biotin was used, resuspend the$ L3 a! o- w$ v0 Z
cell pellet in 100 μL of buffer, add 10 μL of anti-biotin antibody4 s; M( _' Y& c; }
(Anti-Biotin-FITC # 130-090-857, Anti-Biotin-PE # 130-090-3 V9 a; Y$ z: k* c( d8 o( \
756, or Anti-Biotin-APC # 130-090-856), and continue as
* j" g, W2 r/ P1 Sdescribed in steps 4 and 5.
7 ]) c. {" d# A: g3 g7. Resuspend cell pellet in a suitable amount of buffer for analysis2 i, @! Q* F1 E
by flow cytometry or fluorescence microscopy. |
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发表于 2011-9-23 23:57
