求教：AP染色问题

amosummer

最近进行了一个克隆球染色技术。细胞长在96孔板内，严格按照说明，培养5天后进行染色。本来没孔已经长成7-8个克隆球了，但染完后发现这些球都不见了，细胞也很少，会不会是我们洗的时候操作有问题呢，把克隆球都给吸走了。有做过的同志们请告诉我如何改进实验，谢谢了！

amosummer

1，吸取培养基
' B* A* F' e& m. `# I/ Y3 i2，加4%多聚甲醛固定1-2min;
, x6 h  p: ?8 V5 S/ ]2 H3,  用rinse buffer 洗两次；4 Q- C* z( G/ R
4，加入AP染色混合液，暗中室温培养15min;
7 E# c. X/ u2 k3 i5, 吸除染色液，用rinse buffer 洗两次( i# \# e3 l( X/ E5 K& M+ L# v
6，加入PBS，显微镜下观察

amosummer

回复 ghfvcat 的帖子
: q) @* v* T" {4 m9 f/ p  |& A/ w, n. u  l. i
请问在96孔板内一般要养出多少个克隆才进行染色呢？是不是克隆太少的话不太容易染呢？

amosummer

回复 ghfvcat 的帖子* M; m( M3 J+ s0 J
- v8 @* ]$ L. }6 R- L: P9 }- J
药品时间放久了对实验影响大不大呢？

amosummer

回复 ghfvcat 的帖子& R- O: \+ ]. I: F$ Z" S
* o" g/ i# B: Y/ u+ H: u
是millipore的
2 ~2 k% n& u/ Y, A1. Culture ES cells for five days prior to analyzing AP activity, at low to medium density (NOTE: This
% m- e5 r- p" ~7 ]# X/ q3 vtime-period is critical if activity levels of AP needs to be observed. According to our findings, five7 k( G; |" i7 k3 g; z
days of culturing are optimal for good AP stain visualization).. X% x8 Q. [" `* X( g4 X! @
2. On day five, aspirate media and fix ES or EC cells with a fixative (e.g. 4% Paraformaldehyde in9 M. J+ X# W# O' B+ C
PBS) for 1-2 minutes.
) s  p" Q7 _. J+ y: Z; BNote: Do not overfix. Fixing cells longer than 2 minutes will result in the inactivation of alkaline
' Q& W1 |- }+ J, Y3 o; Zphosphatase.: W! P' E! a, b
3. Aspirate fixative and rinse with 1 X Rinse Buffer. DO NOT allow wells to dry.
6 `. B+ ~8 \9 |5 W& m6 E) D+ _0 d' n- k0 J4 X$ z% N
4. Prepare reagents for Alkaline Phosphatase staining as described in “Preparation of Reagents”section.
( B% t$ S- B1 ~( e: k* ]5. Add enough stain solution to cover each well (e.g. 0.5mL for a well of a
6 T7 [# s+ q* t) l24-well plate). Incubate in dark at room temperature for 15 minutes.
7 l3 W: k9 V9 b& S& @6. Aspirate staining solution and rinse wells with 1 X Rinse Buffer. Cover cells with 1 X PBS to
( j5 B: a8 Y1 Y9 Qprevent drying and then count the number of colonies expressing AP (red stem cell colonies),) g6 \: I* u( x6 l* s
versus the number of differentiated colonies (colorless).% u) q$ l" G( n: M$ ^, H0 G5 c7 F
7. AP staining criteria: Greater than 90% of colonies should remain undifferentiated and express alkaline phosphatase in the well containing 103 Units of LIF. P value shall be ³ 0.05.