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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑
^ u' |- h: D$ r) fchangbaihou 发表于 2013-9-3 10:38 
5 ?, K" f3 h: b: \多谢回复，然后呢？怎么用OCT包埋？: }/ }. J8 X. z, R4 ?$ H
请问哪里可以找到protocol啊？
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Preparation of neurospheres for cryostat sectioning and immunocytochemistry:$ R2 K f" J" g/ \+ j% J% s
" n$ R0 x5 U- r# X) {4 ]! ~Procedures have been developed to section intact neurospheres. ' T/ P5 ~ K$ k4 _+ d
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1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.
`" v+ G, [9 p2. 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.$ r, @; W u, j
- s2 w+ Q1 F# s) A, r, LCaution' K& Z( C; B) g- u# U2 n
Centrifugation of the viable neurospheres will alter the shape of the neurospheres. ~( c; b/ d8 }- K
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3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).- ]8 k \/ h* Q/ {% ~6 K) a
4. Allow the neurospheres to settle in the tube.: A. R2 {0 k( D( A4 _& { J
5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS).
@7 e9 ?) n6 J4 f l) y6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.
5 M z% ^9 T- T/ x7. Repeat the rinsing procedure at least 3 times.0 a9 q- ]8 K1 C7 R
Immunocytochemistry may be performed on intact neurospheres at this stage.
; ]0 @; l0 U1 L4 }8 H. v! _ OTIP: Observing the removal of the supernatant at any step under the dissecting scope ensures maximum withdrawal of the fluid without disturbing the cell pellet., @6 V6 s9 c% e8 w$ s; ?) e* u" A! `: K
8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).; a: M' G% u9 Z
9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.' d, H- |; A5 i; D8 ?& @
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)3 o: b$ |& V, K3 S+ D6 A6 k' A
11. Prepare the cryostat chuck with a layer of O.C.T.
: }4 ^2 n0 }1 n! q12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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Caution
) ], n- {3 d3 H- R+ AYou 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.3 ^: I1 Y4 i( S
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
