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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑
; s* P. `$ v2 g/ p$ cchangbaihou 发表于 2013-9-3 10:38  ( h: F$ I, \1 }; C, c
多谢回复，然后呢？怎么用OCT包埋？
* f% W4 S- e% _2 r* Q! b# F请问哪里可以找到protocol啊？
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Preparation of neurospheres for cryostat sectioning and immunocytochemistry:" R( h( s1 F. E, K/ ~; J; G
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Procedures have been developed to section intact neurospheres. 6 o" n, C+ g) g0 G# t
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1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.
9 [0 x8 ~6 x6 H9 B" f, ` `" T2. 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
* H2 O( {9 c, W2 ?( sCentrifugation of the viable neurospheres will alter the shape of the neurospheres.2 Y0 F! @0 R3 {) b+ G9 O
/ \; o9 _8 x; f, E3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).3 H* _- |" ?; S
4. Allow the neurospheres to settle in the tube.: t$ v3 f- [9 }* i( N, j% `2 J
5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS).
6 H. f6 u) B: u4 v( M/ E6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.0 g. Y5 G: M) u4 k
7. Repeat the rinsing procedure at least 3 times.; ?0 S* C- {. ~& ?2 y4 s: o
Immunocytochemistry may be performed on intact neurospheres at this stage.
! {3 d0 @# }4 I* o3 b$ N6 u4 @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.9 G. g! P9 x) }( L m6 U+ Q
8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).1 O- g4 U1 l! A
9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.: O5 y" F+ v" ]2 a# O4 f5 k+ Q* g( R
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)* |# o+ O% h' E+ B( h; a+ v W
11. Prepare the cryostat chuck with a layer of O.C.T.
. u& _5 a4 P* c6 r- {% R( u* [12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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Caution
- @: w3 c+ B3 ~' ]" t% l) c }0 u7 |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.( T# l# W' |2 Y
9 _2 f1 U% k2 W; k) V& I) \1 [13. Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (Sigma cat. no. A3648) coated glass microscope slides.: _0 N8 C! s" P4 W$ O q
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
