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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑
7 A4 F% H. H! Fchangbaihou 发表于 2013-9-3 10:38 
+ R* ~& C' `( X# b5 j; j多谢回复，然后呢？怎么用OCT包埋？
@2 o3 s! h- O请问哪里可以找到protocol啊？
& B, u8 ] n" BPreparation of neurospheres for cryostat sectioning and immunocytochemistry:6 R1 Z- ?& X1 {% v; K I# E" J- k
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Procedures have been developed to section intact neurospheres.
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4 F. q' _3 c1 ^) ?' B1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.
. A( F7 X9 C! o* e5 q* ~2 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.$ C) e( E/ |2 ]5 A5 M
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Caution) n7 j! Z2 [6 P7 Y$ P! R/ U
Centrifugation of the viable neurospheres will alter the shape of the neurospheres.) Q8 p' m; Q) X9 E" N: K
2 I: k2 f& T+ O* j8 q3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).
' Q& V! ?$ @6 d" t* R9 L- x4. Allow the neurospheres to settle in the tube.
5 Z3 X& L1 _) c$ }5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS).
6 e/ J, U6 u3 }& L3 l y! d6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.4 H. O$ I6 [$ [- q; P5 J
7. Repeat the rinsing procedure at least 3 times.
' ]+ h1 s2 _7 s) E+ GImmunocytochemistry may be performed on intact neurospheres at this stage.
# o9 Y0 I& L8 s" w9 c$ |. HTIP: Observing the removal of the supernatant at any step under the dissecting scope ensures maximum withdrawal of the fluid without disturbing the cell pellet.
5 W1 T$ i3 r6 o2 R. ?8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).
% B, N* V4 \! ~7 X4 ?9. Transfer the tube to the fridge and allow the neurospheres to settle overnight." X8 Y1 f; m/ _2 I( d/ U$ k
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)
0 j5 w9 q* Q+ L+ g11. Prepare the cryostat chuck with a layer of O.C.T.
8 {/ N) a* d8 D* T, |: q12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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9 B% {# E) I$ b% `7 V, |7 `3 i) [& |* mCaution$ H. `& _& D% X, V& U$ |8 j
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.
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13. Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (Sigma cat. no. A3648) coated glass microscope slides." w, x/ q* ?' G7 P
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
