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免疫荧光的方法依据你使用的试剂盒各有不同，最好你仔细阅读试剂盒的说明书按照所给程序进行操作。' \: }& ~& ^9 u* {8 {* Y8 K! k6 G6 Q
给你一个方法参考一下，不过是写的荧光双标，与单标的过程基本是类似的只是双标多标了一次。
) c4 e. C8 F( Q; n1 EDouble immunofluorescence staining for BCL-6 and else.* e9 |5 O3 C* [9 H
Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved.
1 U1 G- G. e. U q% {; jUse cytocentrifuged cells or frozen or paraffin dewaxed sections.
. c( Z8 i" g' o/ SPresence of endogenous fluorescent substances may affect your staining.
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! X# `3 s3 _% Y7 yNote: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique.
; y0 x |2 [$ y) ]9 XStart at this section for unfixed or acetone-fixed specimens.
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* `+ a0 h4 b+ H/ X1- Mark the slide in order to recognize the area to be stained and label the slide. 5 T' l0 v$ \+ ?, m6 b
2- Fix in acetone for 10 min at RT [this tep can be omitted]. % J; A$ u: C% [& K6 X5 Q
3- Blot the slides and let them dry for 10 min - 2 hrs. " \. k0 |- i- V: F: ]' H; d
4- Fix in 10% formalin for 10 min in a Coplin Jar.
; D1 b ~3 }, DFrom this point on, don't let dry the slides at any time. 4 w4 N' N# e1 d! Q# m
5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK). B* Q+ m3 `. _( v& o5 W+ C
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Start at this section for dewaxed, antigen retrieved slides
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% Q( L. y0 W0 f* |" G6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash.
- d5 {& B G" r$ W/ ^; D4 ]7- Briefly blot the slides without letting them dry and then apply 3% human serum (health hazard!) or pig serum a s a blocking agent.
* x( d3 M% ~% I0 W3 z4 y" K1 O8- incubate with the blocking for 10 min.
# V5 n5 |! n" m8 v' e9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr. ) J* P$ s2 J- x- o" I
10- Wash twice. 2 Q6 ^, S! w0 P7 e$ S2 k) ~
11- apply a mixture of secondary antibodies (see example below) for 45 min at RT. " O# g, W3 x3 H- _
12- Wash twice.
% _' c' U# k: k. G13- add the mixture of primary antibodies and incubate for 15 min.
, k0 k- S* D9 K. R: j& f14- Wash twice .
) @' ^& ~, m R" k7 d15- add the mixture of secondary antibodies and incubate for 15 min.
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4 w) p0 w; y: M2 L# T1 | T proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19. _% \& u2 \" s* [, u
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16- dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min. 3 V" X1 o: a+ S3 Y+ Z% |) O
17- Wash thrice. 0 ?5 j# h" ]; M0 B1 T2 Y1 V- U7 e4 c2 @
18- Incubate with the avidin for 15 min. 6 l7 k( D- Y6 Q. ]8 c8 G+ o
19- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.
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Example: Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide + mouse anti HA supn 1:100. 7 y A5 i0 w+ F) ^% N& F: X$ @
Secondary antibodies: biotin-conjugated goat anti rabbit (1:200) + TRITC conjugated goat anti-mouse (1:200). Alternatively, biotin-conjugated goat anti mouse (1:200) + goat anti rabbit TRITC (1:200)
1 C$ m) _& ]$ F# \ VAvidin FITC: 1:300
& B. r/ q, l& r8 nTo see the examples click on the thumbnail. 7 ~( W: _; `6 D. P
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发表于 2011-6-8 23:57
