e**f
发帖数: 144 | 1 要做GFAP,O4,Tuj1 triple labeling
GFAP 是rabbit的，O4,Tuj1都是mouse的，请问有没有好的非 rabbit,非mouse的O4或者
Tuj1 抗体可以推荐？
谢谢！
另外，astrocytes-specific marker,除了GFAP，还有别的吗？ |
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s******e
发帖数: 370 | 2 tuj1 is a cytoskeleton protein, not sure if you really need the antigen
retrieval step (citric acid, boiling, etc), usually it's for nuclear
antigens
also are you doing adult or embryonic brains?
tuj1 level decreases in adult CNS but remains high in PNS, try a positive
control with embryonic brain sample |
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n********k
发帖数: 2818 | 3 anyone has good experience with Tuj1 staining, it works for us but with
pretty background, would love to work out a better protocol for one study...
Any tips/informations would be highly appreciated |
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R****e
发帖数: 53 | 4 how do you tell which is or which is not bg for Tuj1?
It is a very good ab. Try low conc. |
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s******e
发帖数: 370 | 5 this shouldn't be very hard, tuj1 is one of the best antibodies
we order it from covance
also check other conditions as well, like 1st/2nd ab conc, species
reactivity, and so on. and have a positive control. |
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a****d
发帖数: 1919 | 6 We use Mouse IgM anti-O4 from hybridoma bank, and Mouse IgG2b Anti Tuj1 from
Sigma. |
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i*********0
发帖数: 915 | 7 我做的EB formation assay，实验的目的是想看看EB 有没有neuronal
differentiation，用Tuj1的抗体来检测。同时，我用oct4的状态（也用是免疫荧光）
来判断细胞的状态（stem cell or differentiated cell）。oct4+ 的细胞，在核周围
可以看到Tuj1的信号，但是oct4-的细胞基本没有Tuj1的信号。
我用的是sigma T2200的Tuj1的抗体，1：200 dilution， cryosection 染色。
oct4用的是santa cruz的。 |
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i*********0
发帖数: 915 | 8 我也觉得奇怪，在我的那个line 的“es” cell中，有Tuj1的表达，但是，一分化，
tuj1就不表达了，但是在分化以后，tuj1在neuron中又开始出现了。 |
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a*****x
发帖数: 901 | 9 其实我还是有疑问。我虽然不是专家，但是也培养过iPS，并derive成neuron.Tuj1的信
号在iPS里是根本看不见的。到neuro progenitor stage 有一些，大概10%左右。sort
NCAM以后继续分化，绝大多数才有Tuj1。 |
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u**********d
发帖数: 573 | 10 是否确定是tuj1的信号？可以做做western看能不能在ES的WCE里检测到条带，及大小是
否合适；
是否其它的ES细胞系也是这样？
如果你确实检测到的是tuj1的信号，我怀疑你的ES疯掉了，比如REST没了，可以看看其
它的neural lineage marker信号。
另外，确实有报道说oct4能在NSC中残留的。 |
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a**p
发帖数: 49 | 11 one way is to increase triton concentration
or inlcude if u hv not
it increase AB penetration and reduce background |
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n********k
发帖数: 2818 | 12 Thanks a lot, folks! Your responses light new hope in my despair heart:)))
Could I have your protocol and also a picture of your staining....
here is what we do: 30 um microtome brain section and focus on Hippocampus:
we mount the section and dry for several hrs and 0.01M(PH6) Citric acid
for 15 min at 95-100, and then
cool down twice in TBS for first time 2-3 min, second time 10 mins.
blocking with 3% DNS plus 0.3% Tween-20 for 1 hr and primary ON in
blocking solution,
(we tried anywh |
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a**p
发帖数: 49 | 13 30 micron meter
well
u can do it in floating sections for staining
with triton rather than tween
staining overnight at 4 degree
TWEEN is too weak
if background too high
reduce concentration of 2nd Ab as well
not necessarily the 1st |
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n********k
发帖数: 2818 | 14 Thanks again...tried free floating methods with virtually no staining at all in all
concentrations..I believe we tried Triton-X100
as well, but will check with my team, we also tried to even wash with 1-5% Triton-X100 afterwards from
anywhere 1 to ON...
BTW, we do lots of staining with many many different Abs but for for some reasons this has been one not
working well with the labs around here( maybe I am asking too much for this staining)...With that, I am really
looking for very specific/firs |
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a*****x
发帖数: 901 | 15 neuron stem cell 应该没有Tuj1吧。真正的ES应该没有信号的 |
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n********k
发帖数: 2818 | 16 同召唤，他是谁呀，那年评的，怎么找不到这个人的说。
前一阵子本来想回的，不过我不做ES细胞，不是很确定，所以想想算了。我说说我的对
这个简单问题的富杂想法哈，ES是个COMPETENT的状态，所以很多基因在ES都有本地表
达，尤其是NUERAL的，有不少表达量还挺高，但可能是完全不相关的功能，比如说NR1
（？），表达量就很高；TUBB3 是一直作为神经元分化的标记，但是其实在干细胞是有
表达的，而且mRNA是不低的，很多神经基因在干细胞比前体细胞表达更高，文献中有，
我自己也看到。还有，我目前用的TUJ1抗体都是有背景，而且不低，也可能是本底表达
，分化的话， 染色会增强很多很多，所以我不看片子，我不相信任何人的照片，太容
易造假了，不过形态可以帮忙确定。简单的说，就是我不知道ES的情况，但是表达不表
达很多时候是相对的。 |
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i*********0
发帖数: 915 | 17 请问一下这个是那一片文献。
我在slide上看到的确实是Tuj1在ES中表达比分化的弱很多，但是相对与其他的一下已
经分化的细胞来说大一点。
NR1 |
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l**********1
发帖数: 5204 | 18 If olfactory epithelium (OE) ES cell,
pls refer one paper:
Wang YZ. et al. (2011)
Canonical Wnt signaling promotes the proliferation and neurogenesis of
peripheral olfactory stem cells during postnatal development and adult
regeneration.
J Cell Sci. 124: 1553-63
Pubmed link:
//www.ncbi.nlm.nih.gov/pubmed/21486944
citation:
Tuj1+ (anti-neuron-specific class III beta-tubulin) neurons
were also located toward the periphery of the flattened sphere (Fig.
5E), and their neurites or axons passed among... 阅读全帖 |
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l**********1
发帖数: 5204 | 19 LZ 的细胞培养过程中 有stress 的话
it might happened. (NB: here stress likes oxgen concentration value)
please refer
Fernandes TG et al. (2010)
Different stages of pluripotency determine distinct patterns of
proliferation, metabolism, and lineage commitment of embryonic stem cells
under hypoxia.
Stem Cell Res. 5: 76-89.
citation:
>beta-tubulin (Tuj1) revealed a slightly higher percentage of
neuronal cells at low oxygen tension that
link:
//www.ncbi.nlm.nih.gov/pubmed/20537975 |
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f*****n
发帖数: 135 | 20 换个神经元的marker看看，比如tuj1，看看是否有相同的现象 |
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d****h
发帖数: 46 | 21 For rat neuron
GABRA1: (Neuron. 1989 Dec;3(6):745-53.A novel alpha subunit in rat brain
GABAA receptors.Khrestchatisky M, MacLennan AJ, Chiang MY, Xu WT, Jackson MB
, Brecha N, Sternini C, Olsen RW, Tobin AJ.)
GABBR2:similar to GABRA1, highly neuron-specific
MAP2:(A Harada, J Teng, Y Takei, K Oguchi, N Hirokawa
MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and
proper PKA signal transduction J Cell Biol, 158 (2002), pp. 541–549)
Tuj1: (J Neurobiol. 1995 May;27(1):26-43.Div... 阅读全帖 |
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j*****9
发帖数: 716 | 22 是确认iPSCs有多能性，就是常说的characterization。通常两种方法都可行，要么送
公司做核型分析（一般实验室做不了，做的了的看不懂的居多，我就看不懂）
karyotyping, 要么做EB再分化成三层：
Ectoderm对应的常用maker是 Tuj1
Mesoderm Smooth muscle actin
Endoderm alpha fetoprotein
然后才分化成你要的特定细胞类型 |
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