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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑 4 r' G; G3 ^ h( O' K; f
changbaihou 发表于 2013-9-3 10:38  / h* h0 T! X" X# S
多谢回复，然后呢？怎么用OCT包埋？
O5 m \; D9 k请问哪里可以找到protocol啊？
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Preparation of neurospheres for cryostat sectioning and immunocytochemistry:* h: _5 W( W3 S! F5 m4 C9 a
0 C5 d" I0 o- Q' [7 y" SProcedures have been developed to section intact neurospheres.
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1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.
}* } {* f2 v% t' [# w2. 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.9 {; |2 [! w8 w- K
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Caution4 l w( w( m+ p5 g! q
Centrifugation of the viable neurospheres will alter the shape of the neurospheres.
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3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).1 ~7 L }( G+ w+ [$ C& H
4. Allow the neurospheres to settle in the tube.
- Q. L% j9 n6 _4 V+ M2 S& G% m' c5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS).
! |( g, V; [+ u$ `; X' [: K# a6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.
; t& O$ ?, C! B; B! ^. V7. Repeat the rinsing procedure at least 3 times.* K" y Y5 C, T; y
Immunocytochemistry may be performed on intact neurospheres at this stage.& B2 B* k& P: I, y
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.
% j# M) d/ x# h# a6 Z$ w' w, ?7 W8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).: y, F; {2 I8 w$ H( m
9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.6 c& m' z2 f: U' y6 }6 s
10. 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)9 w- a5 e) A: U
11. Prepare the cryostat chuck with a layer of O.C.T.0 e8 q* j6 ?( M
12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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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.- g2 z' P. O3 b
' |' L4 } Z! q, X |0 v" B13. Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (Sigma cat. no. A3648) coated glass microscope slides.& C1 H8 K5 }% V2 A# m
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
