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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。
6 g. F- ?" h O- b" |; i* f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。$ q: g9 ?0 S6 Z9 t. |
Double immunofluorescence staining for BCL-6 and else.
) }5 b S/ }" o6 p6 @& bAny type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved.
- C6 n0 H- N2 NUse cytocentrifuged cells or frozen or paraffin dewaxed sections.
9 I' n4 t# @0 D5 F: O$ V. ]Presence 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. 6 E4 m5 I& }9 k! |
Start at this section for unfixed or acetone-fixed specimens. & P' t6 d. y( ^1 F* w" T" |
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1- Mark the slide in order to recognize the area to be stained and label the slide. ; j7 L' Y" o, ?. p+ c& f
2- Fix in acetone for 10 min at RT [this tep can be omitted].
3 H" s$ B. P9 K9 o3- Blot the slides and let them dry for 10 min - 2 hrs.
, _$ t+ g! z" k$ x3 A# m4- Fix in 10% formalin for 10 min in a Coplin Jar.
; w( t) C" X. {: r/ TFrom this point on, don't let dry the slides at any time. ) F7 f) _: S: f6 `# A3 i1 f O
5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK). % g1 b+ ]# `" ] l4 \2 o2 E! B
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Start at this section for dewaxed, antigen retrieved slides ; Q6 k2 m: {: l* ^* h0 E7 v4 r
) i0 M" e: y4 A6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash. & {% n4 ~& `7 _
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.
B! Q% \. v5 Z8 n; j- k8- incubate with the blocking for 10 min. . i% ]7 b6 A% Z4 |# F
9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr.
! a5 b+ t( B3 t& M" i10- Wash twice.
5 [" d" c& l/ Y, C- C- J4 i3 D11- apply a mixture of secondary antibodies (see example below) for 45 min at RT. $ k, C& W& w/ ` L; c# x
12- Wash twice. " [7 _5 k0 R% Y9 w! }
13- add the mixture of primary antibodies and incubate for 15 min. ; X' Y D& \ C% g& c5 s5 w
14- Wash twice . R, P6 O( \- {9 l( L
15- add the mixture of secondary antibodies and incubate for 15 min. & h/ P3 o3 X; P! [
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proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19.% ? ]! d; P6 }; o0 |" U
3 W X5 _* l% E2 n2 \2 @" t* \16- dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min. , `' D0 O/ m5 X) I' K e, h+ R
17- Wash thrice. 2 Q( n- S# C0 P3 H" d, m
18- Incubate with the avidin for 15 min. 2 s. l0 a. l% M# F
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. 3 C* Z- n* u: E! m( P+ z! M1 S7 |
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) % t, G' I/ a) f: K: ~
Avidin FITC: 1:300
2 P6 i' a) H4 E. i; d+ pTo see the examples click on the thumbnail.
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发表于 2011-6-8 23:57
