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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。
S$ J* q3 d: E6 w# Z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。
' A9 H E8 a e. TDouble immunofluorescence staining for BCL-6 and else.
' `2 w1 m: l% S( }+ A1 cAny type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved.
2 y$ s# b* i3 P6 _4 O. ^/ P0 }Use cytocentrifuged cells or frozen or paraffin dewaxed sections.
# U) L' Y& w+ e9 y9 B. FPresence of endogenous fluorescent substances may affect your staining.
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: U! Y- J# G5 Z3 u! S5 P v- [' j% UNote: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique.
% x, U3 L: ^$ b* J l& x2 kStart at this section for unfixed or acetone-fixed specimens. . M$ b8 [4 V0 G8 n
; G. @ W9 I# X8 X' ?) B2 w1 |1- Mark the slide in order to recognize the area to be stained and label the slide. 4 F0 I j z8 y) o: ?; ?
2- Fix in acetone for 10 min at RT [this tep can be omitted].
+ Q' t1 J# i, }3 w& E. B3- Blot the slides and let them dry for 10 min - 2 hrs.
- s: a% X! I9 @. u; r" H4- Fix in 10% formalin for 10 min in a Coplin Jar. 3 K& l B4 q1 }$ {" X" u
From this point on, don't let dry the slides at any time. , d: X1 B/ ^7 f
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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3 ]7 |# {) X/ X* i, A+ {6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash.
" D2 ~9 y& |, A' u( Q5 j3 e+ r B0 f; p7- Briefly blot the slides without letting them dry and then apply 3% human serum (health hazard!) or pig serum a s a blocking agent.
% ?* [* z2 T( ~# s0 ]8- incubate with the blocking for 10 min.
4 `" k2 B! ]% N; h/ O9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr.
. h& Y# P& J. c6 s0 L1 o. v10- Wash twice.
7 G& K+ |# Y: @! U9 ?) r% y5 @11- apply a mixture of secondary antibodies (see example below) for 45 min at RT. 4 u& r" R, W- U4 j/ X0 i
12- Wash twice.
) V2 v/ i4 m' W8 `) p$ J6 K3 w: V" ~13- add the mixture of primary antibodies and incubate for 15 min.
1 A7 v# `& e# x0 n3 B: R14- Wash twice .
# H- z) h1 n u; U0 Z4 o; r9 G, B15- add the mixture of secondary antibodies and incubate for 15 min.
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( s& k) p3 o) M7 p 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.
: F5 L$ Y. ?5 T3 U( y2 q' u: H17- Wash thrice.
! {( R1 y; h3 _0 o18- Incubate with the avidin for 15 min.
9 l6 G7 N9 j4 {, ? v19- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.* R* N2 I* W6 J+ J5 {9 q- Z. u
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Example: Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide + mouse anti HA supn 1:100. 9 T! U3 o6 ^! x3 z- y
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)
5 d" p. X4 q! r: J8 S2 fAvidin FITC: 1:300
- v# g' i+ u/ u1 Y- \To see the examples click on the thumbnail.
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发表于 2011-6-8 23:57
