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标题: 免疫荧光染色步骤 [打印本页]

作者: shanghainese 时间: 2007-5-6 12:02 标题: 免疫荧光染色步骤

Immunofluorescence Method

Frozen Sections
Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at - 80 º C.
Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at - 80 º C until needed.
Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes.
Wash in PBS
   Paraffin Sections

  Procedure for Immunofluorescence Staining

1.    Rinse Sections in washing buffer for 2x2 min.
2.    Primary Antibody (没有标记一抗): incubate sections in primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight. Note

o not rinse sections between serum block and primary antibody incubation.
3.    Rinse in washing buffer for 3x2 min.
4    Secondary Antibody: incubate sections in biotinylated secondary antibody (生物素标记的二抗) (1:500, Vector Labs) in secondary antibody dilution buffer for 30 minutes at room temperature.
5.    Rinse in washing buffer for 3x2 min.
6.    Detection: incubate sections in FITC-Avidin D (1:500, Vector Labs) in PBS for 30 minutes at room temperature. Protecting slides from light starting from this step to the end by covering slides with aluminum foil or black box.
7.    Rinse in washing buffer for 3x2 min.
8.    Counterstain with PI or DAPI if desired for 20-30 minutes at room temperature.
9. Rinse in washing buffer for 3x2 min.
10. Coverslip with Vector aqueous Anti-fade fluorescent mounting medium and seal with nail polish.
11. Store slides in dark at 4 ºC.

作者: iamyueyueyue 时间: 2007-5-15 16:52 标题: 回复 #1 shanghainese 的帖子

学习了，谢谢

作者: 柳玉山 时间: 2007-11-18 19:35 标题: 用丙酮的作用是什么

不知道用丙酮的作用是什么？麻烦您给答复一下好吗？

作者: shanghainese 时间: 2007-11-19 00:05

引用:

原帖由 柳玉山 于 2007-11-18 19:35 发表
不知道用丙酮的作用是什么？麻烦您给答复一下好吗？

fixation

作者: 痞子张 时间: 2007-12-4 09:15

laoshi老师有没有汉语版的啊

作者: shanghainese 时间: 2007-12-4 11:26

没有汉语版。
不过如果你有困难的话，等我有空把它翻过来。

作者: chenxin831125 时间: 2007-12-16 10:22

我们也做免疫组化，不过不会免疫荧光染色，来看看，竟然全是英语，英语不好，不能全看懂。

作者: shanghainese 时间: 2007-12-16 12:28

引用:

原帖由 chenxin831125 于 2007-12-16 10:22 发表
我们也做免疫组化，不过不会免疫荧光染色，来看看，竟然全是英语，英语不好，不能全看懂。

拿本英文字典，一边看一边翻，慢慢就学会了。
其实这个染色步骤（protocol) 是很简单的。
大家有兴趣可以试试。

作者: 方与圆 时间: 2007-12-27 21:04

作者: lencho83 时间: 2008-4-3 16:08

丙酮用什么浓度呢？怎么配制？

作者: shanghainese 时间: 2008-4-4 07:38

引用:

原帖由 lencho83 于 2008-4-3 16:08 发表
丙酮用什么浓度呢？怎么配制？

用纯丙酮。

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