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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑 $ c' H) N/ G. V; B, l
changbaihou 发表于 2013-9-3 10:38  5 W7 o; x8 B! x
多谢回复，然后呢？怎么用OCT包埋？( i, S. P$ ? q. p0 U
请问哪里可以找到protocol啊？
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Preparation of neurospheres for cryostat sectioning and immunocytochemistry:8 @6 A4 H. |# F# t. K; Z5 x* X
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Procedures 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.
8 l8 ?# n5 m/ Z0 s2. 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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Caution2 r3 D% X8 [! {* e% C
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).) U% H y3 R, D$ j$ J3 Y& J6 u
4. Allow the neurospheres to settle in the tube.
/ H5 ?. G0 F, r: d5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS). 1 t; S, |) C2 `2 ?, x
6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.3 b& S* M5 ]3 e/ o) D+ l2 m7 N
7. Repeat the rinsing procedure at least 3 times.* e% I" p9 c0 _
Immunocytochemistry may be performed on intact neurospheres at this stage.
& r0 c F8 k7 u9 v. iTIP: Observing the removal of the supernatant at any step under the dissecting scope ensures maximum withdrawal of the fluid without disturbing the cell pellet.
) Z1 r0 W9 P1 _8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS)., d8 Z" u' E( e8 |. Q
9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.
3 b* B" f# u" l. r& p2 B& q" V10. 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)/ U/ E9 B) Q2 F" C% {8 s* N
11. Prepare the cryostat chuck with a layer of O.C.T.
6 c: Y9 Y+ g) T: a9 O* [12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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Caution
& f* X y9 o1 L; t( R' j: cYou 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.( m! {/ t% r& y, q/ J# 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
