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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑 7 H) F% N5 r p8 A$ C/ [! C
changbaihou 发表于 2013-9-3 10:38  9 l/ i2 ]# _# x) P
多谢回复，然后呢？怎么用OCT包埋？
2 H$ Y B/ A3 ]) d4 m7 e* r请问哪里可以找到protocol啊？
; J, _( j! v% d% T: _Preparation of neurospheres for cryostat sectioning and immunocytochemistry:
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2 b# \4 ?% a0 |6 s4 EProcedures have been developed to section intact neurospheres.
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* A* W6 c- u) a$ [) \9 v: M7 X" H1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.
7 t7 e7 X3 f* [% H7 V% q0 g; j% y2. Allow the neurospheres to settle by gravity (approximately 30 minutes. The time for the neurospheres to settle is dependent on the size of the neurospheres.
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Caution
! _/ q& u$ O' \ @! l, o1 X" ~Centrifugation of the viable neurospheres will alter the shape of the neurospheres.+ O N! e8 `' a* L) S
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3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).
& a$ d6 f+ B- R! K- {$ l4. Allow the neurospheres to settle in the tube.
' o ~0 o% Q# f# V0 C; L z5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS). & d u) E g' s$ B
6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.; L* f8 |' s4 N
7. Repeat the rinsing procedure at least 3 times.
% o5 j+ K( t- Z, A5 b) k) C `Immunocytochemistry may be performed on intact neurospheres at this stage.1 a$ u e2 T! m b' W( h1 ~
TIP: Observing the removal of the supernatant at any step under the dissecting scope ensures maximum withdrawal of the fluid without disturbing the cell pellet." Q, X8 i/ C3 {7 y8 Z$ D' M
8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).
" ^6 l1 v" |2 k6 c/ z( h9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.
, h E9 L2 _5 Q7 H. x10. Remove the 30% sucrose and add embedding medium for frozen tissue specimens for 1 hour (Sakura Tissue-Tek O.C.T. compound, cat. no. 4583)1 a! n# e& y5 G2 b7 y, ?, F! K! @
11. Prepare the cryostat chuck with a layer of O.C.T.# [( }! t0 ^ ^+ w
12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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Caution- [; B% j) p6 X# u
You can lose the neurosphere pellet quickly during the transfer of the pellet from the tube to the cryostat chuck. With a Pasteur pipette or an 1000 μl Eppendorf pipettor (using the blue tips) pre-load a small amount of O.C.T. into the tip and then withdraw the neurosphere pellet into the tip, continuous with the pre-loaded O.C.T. Try not to induce any air bubbles into the O.C.T.: z: K8 p5 c }# F/ O+ T* k% q
) h, L3 j& d( l13. Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (Sigma cat. no. A3648) coated glass microscope slides./ S' C6 r- h+ j% B$ W3 L) D! i/ |
14. Continue with the desired immunocytochemical procedure (non-specific blocking, application of primary antibody etc.). |
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发表于 2013-9-1 07:58
发表于 2013-9-4 10:15
发表于 2013-9-4 14:00
