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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。
5 W" {( V, U5 E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。# x7 P6 h: M4 H& }8 Z7 T
Double immunofluorescence staining for BCL-6 and else.5 y1 J/ o4 O. Q! W+ Q0 n4 ~% g. W
Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved.
: J( @: h7 M, i* T3 P/ JUse cytocentrifuged cells or frozen or paraffin dewaxed sections.
0 x: G: W. q" }& r8 wPresence of endogenous fluorescent substances may affect your staining.
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Note: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique. 1 K1 m$ k# g* K: z) c5 N
Start at this section for unfixed or acetone-fixed specimens. * f- y5 n% Z! n" ]% l
; S2 d2 G1 y% X, q6 k+ @1- Mark the slide in order to recognize the area to be stained and label the slide.
8 H; d3 X, {" ? `2- Fix in acetone for 10 min at RT [this tep can be omitted].
* s) q* x" ~2 Z, j8 L! M7 e+ s3- Blot the slides and let them dry for 10 min - 2 hrs. # \' c8 i! w* T# d( {$ n. }
4- Fix in 10% formalin for 10 min in a Coplin Jar. - x6 N& l- e" Y- C
From this point on, don't let dry the slides at any time.
, @! P+ Y7 B& V8 x2 y0 {5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK).
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Start at this section for dewaxed, antigen retrieved slides
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6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash. 0 p, A, M! |7 J
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.
, g' J( H- i% P# {) W8 u% r8- incubate with the blocking for 10 min. 0 N8 |( ?1 x0 g0 F$ n0 h
9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr. / k K9 N# }8 N+ Q! `/ q
10- Wash twice. 5 j" ~7 L- a8 M! p; n$ Q1 w
11- apply a mixture of secondary antibodies (see example below) for 45 min at RT.
3 v( A' g/ F# k5 e12- Wash twice.
3 f! k4 z/ c+ D) k" `$ J13- add the mixture of primary antibodies and incubate for 15 min.
' |8 c5 r2 w5 f1 I4 r0 R14- Wash twice .
- r, L* F$ G w9 V* \! z15- add the mixture of secondary antibodies and incubate for 15 min.
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proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19.
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16- dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min.
3 Y8 i2 v: x* u- O6 h1 m17- Wash thrice. 8 @% n% \- c' r; X
18- Incubate with the avidin for 15 min. + V9 H U, A. n1 L, G6 O
19- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount./ n ]9 u- Y/ @/ w6 I: [0 I4 k
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Example: Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide + mouse anti HA supn 1:100. + q* \1 o$ N5 }; ^1 B0 _ D
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) ) S2 ^, f+ z# a- ~
Avidin FITC: 1:300 0 z9 B5 I; q* ?$ j9 E" `
To see the examples click on the thumbnail.
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发表于 2011-6-8 23:57
