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本帖最后由 changbaihou 于 2013-9-4 12:45 编辑 1 P' _, a7 L$ \
changbaihou 发表于 2013-9-3 10:38 
9 h2 m* l0 F: K/ y* T. V多谢回复，然后呢？怎么用OCT包埋？. Y q, D$ x" |0 f# L
请问哪里可以找到protocol啊？
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, c& }4 l; \# h% ^, m+ \) Z" ^Preparation of neurospheres for cryostat sectioning and immunocytochemistry:& S) r6 G& f: a* o/ l- p' f
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Procedures have been developed to section intact neurospheres.
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6 n |7 ^" I' ]" q1. Transfer the culture medium with neurospheres to a 50 ml polypropylene conical tube.& M2 n2 F; {3 k& i: Q
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.
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' a' I! ]3 I2 d$ y/ j% ^3 SCaution9 G8 [9 p4 W% j4 V6 ]
Centrifugation of the viable neurospheres will alter the shape of the neurospheres.8 n; A; B# P6 O8 E6 A) G
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3. Remove the supernatant, add and mix gently with 2.0 ml of 4.0% phosphate buffered paraformaldehyde (fixative).
7 \; J3 Y, r- X5 W' a% s* \& G7 U4. Allow the neurospheres to settle in the tube., V$ {9 o: i$ }4 L
5. Remove the fixative supernatant with a Pasteur pipette and rinse with 5.0 ml of phosphate buffered saline (PBS).
# _7 V2 m* r: P6 C" |7 X! U$ n3 @; M6. Allow the neurospheres to settle for at least 30 minutes before removal of the PBS.
1 n, j' p8 T$ }- U7. Repeat the rinsing procedure at least 3 times.
% Q* h( x$ r$ P2 L2 d3 S" OImmunocytochemistry may be performed on intact neurospheres at this stage./ f: s3 W& U4 a' e% ?
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.. n- R8 h3 ]; z0 Q
8. Remove the final PBS rinse and add 5.0 ml of 30% sucrose (dissolved in 0.1M PBS)." s$ x2 N& L. ^% y, h) }
9. Transfer the tube to the fridge and allow the neurospheres to settle overnight.
0 _7 y+ I5 v4 c3 h. 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), F; G) y( g y, U: P' Y! S+ I) d( W
11. Prepare the cryostat chuck with a layer of O.C.T.- h6 W6 l; |9 \4 _
12. Transfer the neurosphere pellet to the O.C.T. layer on the cryostat chuck.
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& l1 i3 b `5 t" DCaution+ E3 x& c2 W8 R; E# ^0 w; q% R
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." u0 Q H9 p4 N% \# Z# h$ ^$ [: p/ Y
' N4 s* X* m* d13. Cut sections at desired thickness and collect on APTEX (3-Aminopropyl) triethoxy-silane) (Sigma cat. no. A3648) coated glass microscope slides.8 Y1 D" r) W; i5 Z
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
