Redian新闻
>
mouse LN immunostaining 的问题
avatar
mouse LN immunostaining 的问题# Biology - 生物学
p*i
1
幼儿园让给万圣节带吃的, 我找了半天找不到那种给小小孩的小杯冰淇淋。
avatar
f*c
2
2天了,仍然查不到. AP renew. Thanks.
avatar
p*e
3
用F4/80染淋巴巨噬细胞,用LYVE-1染淋巴上皮细胞,为什么这两种抗体总是
colocalize?谢谢
avatar
r*f
4
grocery store有一小杯哈根打死一块多的那种,对小小孩来说量挺不少的
不过,你打算怎么保温呀,一定要带这个吗
哦,对了,这种sorbet里面也是小杯子的,超市也都有卖
http://www.luigis.com/home1.aspx
颜色口味估计也投合小朋友的爱好

【在 p*i 的大作中提到】
: 幼儿园让给万圣节带吃的, 我找了半天找不到那种给小小孩的小杯冰淇淋。
avatar
w*n
5
LYVY1 是染淋巴管的吧?

【在 p****e 的大作中提到】
: 用F4/80染淋巴巨噬细胞,用LYVE-1染淋巴上皮细胞,为什么这两种抗体总是
: colocalize?谢谢

avatar
z*c
6
问这个问题的应该直接拖出去打 :)
Kroger有那种非常小杯的。
avatar
p*t
7
Change the antibody to LYVE-1 eFluor660 from eBioscience.
There must be some problem of your ab.

【在 p****e 的大作中提到】
: 用F4/80染淋巴巨噬细胞,用LYVE-1染淋巴上皮细胞,为什么这两种抗体总是
: colocalize?谢谢

avatar
p*i
8
老师让买一包12杯的,我怎么到处找不到这种规格的呀。

【在 r*f 的大作中提到】
: grocery store有一小杯哈根打死一块多的那种,对小小孩来说量挺不少的
: 不过,你打算怎么保温呀,一定要带这个吗
: 哦,对了,这种sorbet里面也是小杯子的,超市也都有卖
: http://www.luigis.com/home1.aspx
: 颜色口味估计也投合小朋友的爱好

avatar
z*6
9
我怎么记着inflammatory macrophage是表达lyve-1的,我不是专家,隔壁实验室天天
做,听说的...
avatar
p*i
10
我们这儿没有KROGER,我已经逛过三四个超市了,没有那种小小杯的。

【在 z**c 的大作中提到】
: 问这个问题的应该直接拖出去打 :)
: Kroger有那种非常小杯的。

avatar
p*e
11
Good, thanks.
Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by
stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound
healing tissue in vivo and in bone marrow cultures in vitro: implications
for the assessment of lymphangiogenesis. J Pathol. 209:67–77.

【在 z*******6 的大作中提到】
: 我怎么记着inflammatory macrophage是表达lyve-1的,我不是专家,隔壁实验室天天
: 做,听说的...

avatar
vn
12
你这个是买ice cream还是光买cup呀 cup哪里都有卖的呀
avatar
s*9
13
生物版讨论起千青、裙带、争authorship叽里哇啦的上万人看贴,上百人留言讨论。讨
论起真正的生物了,3个人留言,2个外行(不是专家),一个上来什么也不分析就要重
新买抗体。
我也不是专家,专家都在讨论Cui J和内幕,我贡献一下自己的2 cents。
你的staining 肯定有问题。Lyve1只表达在lymphatic endothelium上,不在
macrophage上表达。你如果把图贴出来,可能大家还能帮助你。没有图,我只能猜测有
2个可能:
1)你的F4/80antibody不好。F4/80 antibody有2个clone,一个是CI:A3-1,一个是BM8
。8M8 fix的条件比较tricky,不容易在section 上stain,FACS很好用。如果你用的是
8M8,建议改用CI:A3-1,或者仔细摸一下tissue fixation的时间。Lyve1只有一个
clone (ALY7 ),很亮也很好用。
2)即使你用错了F4/80的antibody,也不应该2个antibody co-localize,LN section
最容易出现non-specific binding的地方就是它的外周(subcapsular sinus),它通
常都粘着fat,取LN的时候务必取干净了。我觉得,有可能你的staining有问题,2个
antibody都没有亮。出来的都是外周的一圈non-specific binding。

【在 z*******6 的大作中提到】
: 我怎么记着inflammatory macrophage是表达lyve-1的,我不是专家,隔壁实验室天天
: 做,听说的...

avatar
p*i
14
I think it is ice cream in cups.

【在 vn 的大作中提到】
: 你这个是买ice cream还是光买cup呀 cup哪里都有卖的呀
avatar
o*d
15
学习了。

BM8

【在 s****9 的大作中提到】
: 生物版讨论起千青、裙带、争authorship叽里哇啦的上万人看贴,上百人留言讨论。讨
: 论起真正的生物了,3个人留言,2个外行(不是专家),一个上来什么也不分析就要重
: 新买抗体。
: 我也不是专家,专家都在讨论Cui J和内幕,我贡献一下自己的2 cents。
: 你的staining 肯定有问题。Lyve1只表达在lymphatic endothelium上,不在
: macrophage上表达。你如果把图贴出来,可能大家还能帮助你。没有图,我只能猜测有
: 2个可能:
: 1)你的F4/80antibody不好。F4/80 antibody有2个clone,一个是CI:A3-1,一个是BM8
: 。8M8 fix的条件比较tricky,不容易在section 上stain,FACS很好用。如果你用的是
: 8M8,建议改用CI:A3-1,或者仔细摸一下tissue fixation的时间。Lyve1只有一个

avatar
l*a
16
那你可以去问问老师,看她有没有推荐的店。我在local的grocery store看到过12杯一
盒的,就是italy ice cream

【在 p*i 的大作中提到】
: 老师让买一包12杯的,我怎么到处找不到这种规格的呀。
avatar
s*9
17
I am not sure about tumor microenvironment. In Lymph node (naive or
immunized mice), Lyve1 and F4/80 positive cells are in different regions.
Lyve1 is expressed in subcapsular region while F4/80 is in medullary region.

【在 p****e 的大作中提到】
: Good, thanks.
: Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by
: stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound
: healing tissue in vivo and in bone marrow cultures in vitro: implications
: for the assessment of lymphangiogenesis. J Pathol. 209:67–77.

avatar
vn
18
你还是问问老师吧
这边一大罐ice cream + 12cup倒是蛮常见的

【在 p*i 的大作中提到】
: I think it is ice cream in cups.
avatar
h*y
19
收藏了。多谢。

BM8

【在 s****9 的大作中提到】
: 生物版讨论起千青、裙带、争authorship叽里哇啦的上万人看贴,上百人留言讨论。讨
: 论起真正的生物了,3个人留言,2个外行(不是专家),一个上来什么也不分析就要重
: 新买抗体。
: 我也不是专家,专家都在讨论Cui J和内幕,我贡献一下自己的2 cents。
: 你的staining 肯定有问题。Lyve1只表达在lymphatic endothelium上,不在
: macrophage上表达。你如果把图贴出来,可能大家还能帮助你。没有图,我只能猜测有
: 2个可能:
: 1)你的F4/80antibody不好。F4/80 antibody有2个clone,一个是CI:A3-1,一个是BM8
: 。8M8 fix的条件比较tricky,不容易在section 上stain,FACS很好用。如果你用的是
: 8M8,建议改用CI:A3-1,或者仔细摸一下tissue fixation的时间。Lyve1只有一个

avatar
p*e
20
谢谢了。我给老鼠打了CpG ODN,淋巴结有平时3-4倍大,所以Macrophage 应该是在
inflammatory状态下,抗体的clone我从来没注意,回头看一下(我本行不是学生物的
)。我的F4/80和Lyve-1荧光大都在subcapsular sinus area,图明天贴上来。多谢各
位。

region.

【在 s****9 的大作中提到】
: I am not sure about tumor microenvironment. In Lymph node (naive or
: immunized mice), Lyve1 and F4/80 positive cells are in different regions.
: Lyve1 is expressed in subcapsular region while F4/80 is in medullary region.

avatar
s*9
21
F4/80 macrophage在medullary region,subcapsular macophage表达Moma1,不表达F4
/80。如果你的inflammatory macrophage 都表达了了lyve1(觉得不太可能),那所有
的lymphatic endothlial cell必须也要表达F4/80 2个抗体才能co-localize。

【在 p****e 的大作中提到】
: 谢谢了。我给老鼠打了CpG ODN,淋巴结有平时3-4倍大,所以Macrophage 应该是在
: inflammatory状态下,抗体的clone我从来没注意,回头看一下(我本行不是学生物的
: )。我的F4/80和Lyve-1荧光大都在subcapsular sinus area,图明天贴上来。多谢各
: 位。
:
: region.

avatar
p*t
22
We are talking about science here. Not necessary to comment on others.
LZ did not give much information of his experiment, so what you listed
in below is also pure speculation.
Here I attach a pic what I stained with LYVE-1 in LN,
which is also inflammatory.
The red signal is LYVE-1, blue is B cell region.
Fat should not be problem in isolating LN.
I agree with you that there might be problem of specific staining.
I assume that in the LN of his model, here might be expansion of
both macrophages and lyymphatics. With DAPI staing under a higher
magnification, he can differentiate macrophages and lymphatics
more clearly.
FACS staining to understand the percentage of F4/80 positive macrophages
compared with control would also give some information.
---------
发信人: spt009 (cytokine), 信区: Biology
标 题: Re: mouse LN immunostaining 的问题
发信站: BBS 未名空间站 (Thu Dec 6 20:16:07 2012, 美东)
生物版讨论起千青、裙带、争authorship叽里哇啦的上万人看贴,上百人留言讨论。讨
论起真正的生物了,3个人留言,2个外行(不是专家),一个上来什么也不分析就要重
新买抗体。
我也不是专家,专家都在讨论Cui J和内幕,我贡献一下自己的2 cents。
你的staining 肯定有问题。Lyve1只表达在lymphatic endothelium上,不在
macrophage上表达。你如果把图贴出来,可能大家还能帮助你。没有图,我只能猜测有
2个可能:
1)你的F4/80antibody不好。F4/80 antibody有2个clone,一个是CI:A3-1,一个是BM8
。8M8 fix的条件比较tricky,不容易在section 上stain,FACS很好用。如果你用的是
8M8,建议改用CI:A3-1,或者仔细摸一下tissue fixation的时间。Lyve1只有一个
clone (ALY7 ),很亮也很好用。
2)即使你用错了F4/80的antibody,也不应该2个antibody co-localize,LN section
最容易出现non-specific binding的地方就是它的外周(subcapsular sinus),它通
常都粘着fat,取LN的时候务必取干净了。我觉得,有可能你的staining有问题,2个
antibody都没有亮。出来的都是外周的一圈non-specific binding
avatar
p*t
23
Please read this paper, you are misleading others.
-----------J Innate Immun. 2012;4(5-6):424-36. doi: 10.1159/000337007. Epub
2012 Apr 4.
Lymph node macrophages.
Gray EE, Cyster JG.
Source
Howard Hughes Medical Institute and Department of Microbiology and
Immunology, University of California San Francisco, San Francisco, CA 94143-
0414, USA.
Abstract
Lymph node (LN) macrophages have long been known for their efficient uptake
of lymph-borne antigens. A convergence of studies on innate and adaptive
immune responses has led to exciting recent advances in understanding their
more specialized properties: presenting antigens to B cells, dendritic cells
and T cells, producing trophic factors and cytokines, and, remarkably,
being permissive for viral infection, a property critical for mounting anti-
viral responses. LN macrophages have been traditionally divided into subsets
based on their subcapsular sinus and medullary locations. Here, we classify
LN macrophages into three subsets: subcapsular sinus macrophages, medullary
sinus macrophages and medullary cord macrophages. We review the literature
regarding the roles of these cells in innate and adaptive immune responses
and requirements for their development. We also discuss challenges
associated with their purification as well as the existence of additional
heterogeneity among LN macrophages.

region.

【在 s****9 的大作中提到】
: I am not sure about tumor microenvironment. In Lymph node (naive or
: immunized mice), Lyve1 and F4/80 positive cells are in different regions.
: Lyve1 is expressed in subcapsular region while F4/80 is in medullary region.

avatar
p*e
24
thanks a lot everyone, here are the images.

Epub
94143-

【在 p**t 的大作中提到】
: Please read this paper, you are misleading others.
: -----------J Innate Immun. 2012;4(5-6):424-36. doi: 10.1159/000337007. Epub
: 2012 Apr 4.
: Lymph node macrophages.
: Gray EE, Cyster JG.
: Source
: Howard Hughes Medical Institute and Department of Microbiology and
: Immunology, University of California San Francisco, San Francisco, CA 94143-
: 0414, USA.
: Abstract

avatar
p*t
25
F4/80 staining is non-specific.
Lyve-1 staining can be better.
Have a look at the attachment, which is from the paper of
Michael C. Carroll 2010 on Immunity. Blue signal is F4/80,
green is CD169. Blue signal means F4/80 positive medullary
macrophage. Although here is no higher magnification, the signal
is so different with yours.
You can also check Jason Cyster's Nature Immunology paper to see
how the signal of F4/80 in their hands. Carefully check your
abs for histology. The problem should be the abs you used.
Good luck!

【在 p****e 的大作中提到】
: thanks a lot everyone, here are the images.
:
: Epub
: 94143-

avatar
p*t
26
btw, in the 1st pic, you should decrease the number of mosaics.
The pic is too small, which can not tell you much information.
10x objective would be better.

【在 p****e 的大作中提到】
: thanks a lot everyone, here are the images.
:
: Epub
: 94143-

avatar
p*e
27
Thanks, I attached the image by Jason Cyster Nature Immunology, it appeared
to me the F4/80 pattern was similar to my image.
Thanks and I learned a lot.

【在 p**t 的大作中提到】
: F4/80 staining is non-specific.
: Lyve-1 staining can be better.
: Have a look at the attachment, which is from the paper of
: Michael C. Carroll 2010 on Immunity. Blue signal is F4/80,
: green is CD169. Blue signal means F4/80 positive medullary
: macrophage. Although here is no higher magnification, the signal
: is so different with yours.
: You can also check Jason Cyster's Nature Immunology paper to see
: how the signal of F4/80 in their hands. Carefully check your
: abs for histology. The problem should be the abs you used.

avatar
s*9
28
Orientation is different. It is hard to tell. It seems that the Lyve1
staining is good.

appeared

【在 p****e 的大作中提到】
: Thanks, I attached the image by Jason Cyster Nature Immunology, it appeared
: to me the F4/80 pattern was similar to my image.
: Thanks and I learned a lot.

相关阅读
logo
联系我们隐私协议©2024 redian.news
Redian新闻
Redian.news刊载任何文章,不代表同意其说法或描述,仅为提供更多信息,也不构成任何建议。文章信息的合法性及真实性由其作者负责,与Redian.news及其运营公司无关。欢迎投稿,如发现稿件侵权,或作者不愿在本网发表文章,请版权拥有者通知本网处理。