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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 E0 R% w& D! K' Y3 ^ ^7 Y, v
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。
4 T0 k" N; y( {Double immunofluorescence staining for BCL-6 and else.
3 m3 a1 C7 f( yAny type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved. ' v3 e. K& u+ A" q8 t
Use cytocentrifuged cells or frozen or paraffin dewaxed sections.
3 e) p3 c) {$ f6 [- y8 I x6 kPresence of endogenous fluorescent substances may affect your staining. & [7 w* }! y3 y/ f
8 {' ` K& K! D# _# B( ]5 L6 Q( T, eNote: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique.
' Z- C; B- H6 F: M! ]5 [Start at this section for unfixed or acetone-fixed specimens.
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& E% T) Q$ c) k7 i; w5 `- U. Q1- Mark the slide in order to recognize the area to be stained and label the slide. . U. n, N! p! Y- [4 D8 I% H6 ^
2- Fix in acetone for 10 min at RT [this tep can be omitted].
# w- w5 O7 g" j: v; f1 S D: z) g3- Blot the slides and let them dry for 10 min - 2 hrs.
7 \1 }& \7 `5 N+ c( Q' H4- Fix in 10% formalin for 10 min in a Coplin Jar.
- I) x2 F% L% WFrom this point on, don't let dry the slides at any time.
) v3 W3 W3 J/ D+ g) M4 k( _5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK). * [" Q! Z. d6 g9 p+ @
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Start at this section for dewaxed, antigen retrieved slides 4 Q9 E5 \3 ?/ E: _ l! q+ n
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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. 8 f* v' C* b7 }5 W# L5 Z
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.( |) H) ?6 ~5 v0 O! i
8- incubate with the blocking for 10 min. & Q: h# |. Q+ _0 }2 I2 g
9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr.
2 g n' H J m: O% _! o% F10- Wash twice. + G7 f0 _$ n1 W- d9 I5 p! k
11- apply a mixture of secondary antibodies (see example below) for 45 min at RT. % ^- U* H3 Q2 j# T) I) A
12- Wash twice. * W6 h. g+ ]4 b4 y" }* R* q+ L
13- add the mixture of primary antibodies and incubate for 15 min.
8 s( q Z3 T$ V3 v- Q& J: r14- Wash twice .
( j' q) \. Z0 |* S15- add the mixture of secondary antibodies and incubate for 15 min.
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* e8 u. `5 T0 d* g# G 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.
' F" h* S% A: Q, U5 I; f! G17- Wash thrice. 9 ]7 R* J/ l/ c2 Y% r
18- Incubate with the avidin for 15 min.
- ?& k/ @* H. V0 T- k0 a# h! B19- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.1 Z; _8 n2 ^* F9 M3 i* }/ U$ X
+ o( G/ W: ?+ B1 P; \! {Example: Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide + mouse anti HA supn 1:100.
/ d; E$ s8 v3 @, |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) 6 O4 m$ u6 c, v2 F. k9 r6 @0 R
Avidin FITC: 1:300
2 v0 R1 P+ J# t# K4 J3 pTo see the examples click on the thumbnail. 7 j9 C/ Y0 K2 w, Q. G% J% H3 Q
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发表于 2011-6-8 23:57
