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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑
8 I% I. K3 t- zchangbaihou 发表于 2013-9-3 10:38 
6 L9 p9 |4 x. a/ R! e: J4 l0 a. V多谢回复，然后呢？怎么用OCT包埋？
$ N' C3 K( {0 O( l0 m请问哪里可以找到protocol啊？
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Preparation of neurospheres for cryostat sectioning and immunocytochemistry:0 L2 j: n3 Q- ]# d3 S
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Procedures have been developed to section intact neurospheres.
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1 Z% E/ C9 p l1 d* b1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.
. i3 ?$ c* y$ ?# ]2. 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.7 N. I* x& ?5 B: a/ v M7 F
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Caution
+ E( O; G" t/ P: o, mCentrifugation of the viable neurospheres will alter the shape of the neurospheres.
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' C0 [0 J: N. d# T- M3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).: p( {6 L2 ]1 u0 H
4. Allow the neurospheres to settle in the tube.# [6 [5 c- A1 {4 L0 G: `& V
5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS). 6 E( v% ~) x4 `! E0 E$ Y% H7 P; L$ n
6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.
7 f6 {4 f! s0 [) p7. Repeat the rinsing procedure at least 3 times.
- g2 V0 t8 ?' ~% _Immunocytochemistry may be performed on intact neurospheres at this stage.
8 W8 ` q' K" k, _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.
4 h/ [* f5 p) B; H8 |" I8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS).: k3 j9 M! S2 y, B8 R) ~9 b
9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.
/ L) Y0 y/ U% R6 ], ^+ @2 Y10. 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)
& V+ H# J+ H M" M11. Prepare the cryostat chuck with a layer of O.C.T." n. z9 i4 k) A& w8 j
12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.- ^! B4 l! j/ f' B$ h
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Caution
# B/ q+ l/ @$ sYou 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.) n9 O# e3 @' U3 d! O$ u9 O: g3 g
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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.
) o4 Y. R# n# c1 [2 _0 [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
