f***4
发帖数: 886 | 1 其实是瓜分钱的问题，建议wellcome以后投钱的方向
他给的建议是
iPS（iN),找不同神经元的转录因子
人胚胎表达库(抗体，in situ)
免费共享资源
病人库 |
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p*****m
发帖数: 7030 | 2 这些都挺靠谱的啊 而且也都是大科学的样子了 也不知道他那么反感omics干啥 呵呵 他
这个抗体库in situ atalas和iPS库也都是omics了的说。。 |
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y*****o
发帖数: 1011 | 3 刚做了BrdU，用20ug/ml prok消化10min，效果很好。
现在要开始做TUNEL了（同一咱tissue），用的是Roche的in situ cell death detection kit，里面提到15ug/ml proK在25-37度下5-30分钟。
想知道如果知道了brdu中，用20ug/ml prok消化10min可以的话，在TUNEL中也用这个条
件行吗（20ug/ml会不会太harsh）？还是试比如15ug/ml proK 37度下 15-20min？
我觉得20ug/ml prok消化10min 应该可以，不过实验室的人不是很同意，说还是用15ug
/ml.。
新手，请指点一下吧，谢谢！ |
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S*****s
发帖数: 287 | 4 信号比较弱。建议你试试 Perkin Elmer 的 TSA kit。 |
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c*******l
发帖数: 54 | 6 谢谢大家的建议！准备试试Roche DIG+Perkin Elmer TSA
同位素信号肯定是强的，不过很多人都不愿用吧，我也是。 |
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D****g
发帖数: 275 | 11 如果是AP染色的话，TRY酒精，注意掌握好时间，脱色很快。 |
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f******u
发帖数: 9 | 12 GENERAL SUMMARY
A postdoctoral position is available immediately in the laboratory of Dr.
Christopher Bartlett in the Battelle Center for Mathematical Medicine to
perform neurogenetic studies correlating genetic variation to gene
expression phenotypes from rodent and human brain tissue samples. Essential
methodologies include using multiplex PCR, RT-PCR, quantitative PCR, tissue
histology, in situ hybridization and immunohistochemistry methods interfaced
microscopy data collection. Experience wi |
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w******y
发帖数: 2504 | 14 Could any friend do me a favor sending a copy of the fllowing paper to my
email: p***********[email protected]? Thanks so much.
Ionotropic glutamate-receptor gene expression in hypothalamus: localization
of AMPA, kainate, and NMDA receptor RNA with in situ hybridization.
van den Pol AN, Hermans-Borgmeyer I, Hofer M, Ghosh P, Heinemann S.
J Comp Neurol. 1994 May 15;343(3):428-44. |
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c*********r
发帖数: 1312 | 15 顶一下，同问。
只不过我想学习一下统计Fluorescent in situ Hybridization的点状物的分布。 |
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y******8
发帖数: 1764 | 17 wow, this would require sub-nm resolution. Could any in situ
technique achieve this resolution? |
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y******8
发帖数: 1764 | 18 It is a very cool technique. However, I don't think this can be applied as
in situ imaging method. |
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m******5
发帖数: 1383 | 19 小弟刚从生化转到发育领域玩
目前正在做一个Homeodomain protein,查10年来的相关的发育领域的文献（DB,
development）非常奇怪地发现都只有in situ 检测蛋白表达和分布，没有免疫组化，
也没有western
我试了市面上所有commercial抗体，也都不能在组织里检测到（western 和免疫组化）
作为阳性对照，转293 overexpress的蛋白检测起来都是没问题的
PS ：我做组织裂解液是用RIPA+超声打散的办法（组织在RIPA buffer里沉淀后直接用
超声打匀，反复三次），理论上也应该没问题？
求指教！
另外还有一个好奇的地方：很多做发育的文章也都只用ｉｎ ｓｉｔｕ来说明问题，这
是什么原因呢？ 是因为组织里免疫检测不到很正常？
另外，没有蛋白水平的检测能说明问题么？ 比如可能蛋白质已经被truncate 了 ，或
者蛋白质aggregate了 ，或者被调控降解了 ，这些都是不能用简单的insitu来证明的
吧？ |
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k****u
发帖数: 3454 | 21 哦，了解
但是这个non invasiveness和real time似乎比较先进。。。
我知道imaging mass spec是可以有比较好的compound identification，但是不可以
real time, in situ...
spectrum. |
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l*****a
发帖数: 1431 | 22 We have patient lymphblast cell line and want to see if some mRNA are stuck
in the nucleus. our plan is to In situ hybridyzation using this cell line.
But the problem is how to fix the cells on slides?
Any suggestion? Thanks a lot |
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w******e
发帖数: 1187 | 23 最好不需要fluorescence，或者在性能方面，能利用sandwich mechanism提高
specificity的。
多谢！ |
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m******5
发帖数: 1383 | 25 我有个蛋白，已知有两个isoform,A form是全长，B form是trancated 的Aform
请问要如何设计探针tell them apart呢？它们的中间片段并没有区别 |
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s******s
发帖数: 13035 | 26 没做过。不过我猜用LNA，PNA这类探针，一个设计在
truncation里面，一个设计跨在两边 |
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w******e
发帖数: 1187 | 27 没理解。用A有B没有的部分做antigen不行吗？ |
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b*********l
发帖数: 490 | 28 My in situ doesn't work at all.
I run out the dig labeled RNA probe in gel and it looks pretty good. Then I
use 100ng-400ng in 200ul hyb mix in a trial experiment, both the Roche AP (1
antisense and sense probes. For NBT/BCIP staining, no blue color showed up.
I am wondering if somebody could give me some suggestions. Thank you very
much. |
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a********k
发帖数: 2273 | 29 2 is very important for in situ. For the first point, you need a positive
control probe that labels your specific tissue as a good control, those
published by other labs should work for you. |
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a******n
发帖数: 392 | 30 根据我做了几年的 in situ 的经验，不需要DEPC处理的水，任何一个步骤都不需要。4
％ PFA 固定24小时，样品里的
mRNA不用担心被降解。
been |
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M*****n
发帖数: 16729 | 31 it is already better to over-think it than under-think it.
try every way you can to make sure in situ works. then you can be lazy and
save some steps later.
been |
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W*******a
发帖数: 1769 | 32 You need to understand how Illumina seq works. Two ends of the
library molecule anneal to two oligos on the slide, forming
a bridge, and a PCR is performed in situ to amplify this molecule
into a cluster. The efficiency and consistence of cluster forming
is limited by the size of the insert, you can't have very long
product (>1kb)
classical pair-ends are generated directly from short inserts
usually less than 1kb, so no special library preparation method is
required.
For mate-paired, the t... 阅读全帖 |
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s**l
发帖数: 11983 | 33 http://www.nature.com/nature/journal/v470/n7334/full/nature0981
An early Ediacaran assemblage of macroscopic and morphologically
differentiated eukaryotes
地球上最古老的「高等生命」到底是什么样子?人类已知的高等生命从什么时期开始演
化?英国的《自然》(Nature)杂誌在17日公佈了这一重大发现。
地球上的生命从无到有、从简单到复杂，其中的进化过程相当漫长。古生物学家认
为，在现今生物圈中，包括我们人类在内的所有肉眼可见的生命，几乎都是多细胞宏体
生物，也就是常说的「高等生命」，而多细胞宏体生物的出现是地球生命进化史上极为
重要的事件。
但是学术界一直认为地球没有早于5.8亿年前的「高等生命」，不过中国科学家在
安徽休甯县蓝田镇找到的「蓝田植物群」把高等生命起源向前推进了近4000万年。
「蓝田植物群」就在知名的景点黄山附近，出现在6亿多年前，展现了早期动物大规模
出现前夕的生命景观，是地球早期生命从简单向复杂进化过程中的重要环节。... 阅读全帖 |
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h******y
发帖数: 351 | 34 I asked the same question before on Histonet and other forum but never got a
satisfactory answer. :-(
I was doing in situ hybridization using TBS or PBS and came across another
protocol using Maleic acid. Then I went back and checked the data sheet
coming with the anti-DIG-AP antibody (Roche), and found that they suggested
using Maleic acid based buffer for "membrane application" and Tris-based
buffer for "other application".
Maleic acid has less buffering capacity than Tris in the pH range of 7... 阅读全帖 |
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h*******1
发帖数: 15 | 35 美国西北大学土木与环境工程系Prof. Yun Wang 招收联合培养博士生
Prof. Yun Wang from the Department of Civil and Environmental Engineering at
Northwestern University is seeking for Ph.D. candidates to conduct research
in her lab as exchange students under the joint-training programs sponsored
by China Scholarship Council (CSC). Students with educational background
in Environmental Chemistry, Microbiology, Molecular Biology, or related
majors are encouraged to apply. Successful candidates must have research
experience/i... 阅读全帖 |
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h********n
发帖数: 4079 | 36 我有个Cre-induced tissue specific KO mouse model.
在mammary gland里, 有些细胞会表达Cre, 可能是epithelial cell. 有什么比较方便
的办法可以知道有多少细胞表达了Cre然后完成KO?
IHC for Cre and the KO gene?
or in situ hybridization for the KO gene?
谢谢. |
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s****9
发帖数: 932 | 38 95% Etoh for 30-60 mins. |
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M*****n
发帖数: 16729 | 39 thanks guys,
will try tomorrow. |
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F*****e
发帖数: 182 | 40 泡在PBST（0.1% Tween-20)里，4度放几天，可以降低一些background。 |
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j*****d
发帖数: 787 | 41 too long! try 3 minutes, 6 minutes, 10 minutes..... |
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c*********r
发帖数: 1312 | 42 不好意思可能会让你失望了，Wnt的diffusion和我们的实验体系其实不太相关，所以我
不太了解，具体怎么测量还真不了解，做immunostaining？in situ？GFP tag？
不过Wnt的diffusion和果蝇胚胎发育早期经典的bicoid、hunchback等morphogen的
diffusion有所不同。bicoid、hunchback等好像都是transcription factor，果蝇早期
的胚胎是合胞体，一个细胞膜内N多核，转录因子直接通过扩散可以如何，调控基因表
达；Wnt是ligand，要和细胞表面的受体结合后经过复杂的信号传导后才有beta-
catenin，c-jun等入核调节基因表达或者有细胞骨架的变化，所以Wnt的diffusion应该
还受到受体分布和下游信号传导的制约的。
我们做的海胆胚胎，早期是没有localized Wnt ligand，只有Wnt下游的蛋白，比如
Dishevelled，是localized，但却足以激活并限制Wnt信号通路只在局部进行。稍晚一
些Wnt8表达，加强了这种效果。
另外关于Morphogen diffus... 阅读全帖 |
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D******9
发帖数: 2665 | 43 想比较一下global chromatin condense change， 有好的protocol推荐吗？ 谢谢 |
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n*********m
发帖数: 38 | 44 Look his publication
Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate: new insights
from structural and biochemical studies on human RPE.
Liang W, Ouyang S, Shaw N, Joachimiak A, Zhang R, Liu ZJ.
FASEB J. 2011 Feb;25(2):497-504. Epub 2010 Oct 5.
PMID:
20923965
[PubMed - indexed for MEDLINE]
Related citations
2.
Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate
synthetase by N10-substituted folate analogues.
Wu D, Li Y, Song G, Cheng C, Zhang R, Joac... 阅读全帖 |
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s****9
发帖数: 932 | 45 Thanks. I will have a try of PFA fixation with protein retrieval.
The purpose of the IHC is to in situ visualize the cell type which has the
enzyme expression(located on the ER membrane). My laser capture data
suggests that the enzyme selectively expressed on certain regions but that
regions contains many cell types. IP is not useful for me.
My concern is that the epitope is intramembrane and thus cannot expose out
after fixation.
t
if |
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l**s
发帖数: 55 | 46 try to dissect cartilage alone, also try 55 degrees (for both in situ and
wash) |
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m**********2
发帖数: 57 | 47 This is E15.5 bone tissue. It's difficult to dissect cartilage alone.
I will try the lower temperature first. Thank your suggestion.
Actually, I found the mineral zones in the middle attach firmly after
decalcification, while chondrocytes and hypertrophic chondrocytes are easier
to detach after in situ incubation compared to no decalcification tissues.
The pic is no decalcification tissues. |
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n*****d
发帖数: 82 | 48 随便google了一下, 有好多, 不知道那家比较好.
有用过提供ISH服务的公司嘛? 多谢 |
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m*****n
发帖数: 760 | 49 自己做不行吗？
也不是很难，关键是要probe设计的好。 |
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w********u
发帖数: 5457 | 50 cell? section? embryo?
有时间还是自己做吧，公司其实也都不可靠。
出钱我帮你做也行，^_^！ |
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