l******u 发帖数: 936 | 1
we have modified one commercial kit to get total-RNA with small RNA from
laser microdissected cryosections.
sure, it require RT and pre-amplification
just treat it as usual way as total RNA preparation for microarray.
microRNAs are such short ~22 nucleotide RNA sequences, so they are more
stable than mRNA, some people even do microRNA array from paraffin samples. |
|
z****u 发帖数: 1007 | 2 最近在从paraffin section 改到frozen section来做in situ hybrdization. new
born mice samle after decalcification. 遇到麻烦就是老是掉片子。section不贴。
。过几次solution就掉的差不多了。。谁有经验的?troubleshooting一下。 |
|
y***u 发帖数: 1467 | 3 每个抗体是不一样的,有的抗体不需要antigent unmasking,有的用EDTA,有的用
citrate buffer。vector 的antigen unmasking solution 好像是citrate buffer (
这个你也可以自己配)。你可以查查抗体的说明上有没有相应的信息,或是看看其他人
用这个抗体用的是什么类型的buffer。都没有信息的话,就自己拿几个片子做预实验了。
我觉得跟paraffin 还是cryosetion 关系不大,跟抗体自身关系大。 |
|
g***y 发帖数: 201 | 4 Ubiquitin(P4D1) #3936 from cell signaling
works very well on PFA fixed paraffin sections |
|
|
g***s 发帖数: 39 | 6 机器应该有个专门process brain的program的吧,我们用的机器每个步骤是50分钟。
3 |
|
D******9 发帖数: 2665 | 7 I have to do some immunohistochemical staining on paraffin-embedded
sections. Thx |
|
b**c 发帖数: 161 | 8 "A Novel Proximity Assay for the Detection of Proteins and Protein Complexes
Homodimerization in Formalin-fixed, Paraffin-Embedded Cell Lines and Breast
Cancer Tissue"
Diagnostic Molecular Pathology:
March 2009 - Volume 18 - Issue 1 - pp 11-21
My email address: d********[email protected]
Thanks so much! |
|
|
m*n 发帖数: 695 | 10 玻片置柠檬酸缓冲液中,微波炉先大火到沸腾然后小火维持将沸不沸的水平十分钟,自
然冷却 |
|
s******s 发帖数: 13035 | 11 没问题,无穷这种FFPE“ formalin-fixed, paraffin-embedded tissue”
的kit,比如qiagen rneasy ffpe kit |
|
M*****e 发帖数: 279 | 12 If you are interested in cell proliferation, phosphorylated Histone H3 S10
antibody is better.
Ki67 is not M-phase specific. Histone H3 pS10 is relatively M-phase specific.
You can use 1:50 to 1:100 for Histone H3 pS10 on Formalin fixed paraffin
embedded tissue section. You can recycle the antibody (add sodium azide) and
re-use it later. |
|
h******u 发帖数: 602 | 13 想看一下一基因是否有突变,手头只有石腊包埋的组织。有没有公司提供类似的测序服
务?
谢谢了!!! |
|
X***n 发帖数: 366 | 14 Try toluene and serial ethanol to remove paraffin and rehydration.
Digestion at 60oC with proteinase K to release RNA and protect from RNase
degradation. |
|
D*a 发帖数: 6830 | 15 white adipose tissue解剖过一次,现在想看看Brown adipose tissue,谁有protocol
或者图示之类的阿?
还有随后想做IHC,用paraffin包埋的时候有什么注意事项么?这玩意儿粘乎乎的,过
了pfa, ETOH, xylene之后该是什么样的?... |
|
l********1 发帖数: 54 | 16 Thanks! I'll try to stain p16, another marker for senescence. |
|
p******6 发帖数: 410 | 17 跟贴同问一下,那些是比较公认的标准senescence marker,似乎SA-B-Gal是个金标准,
不用它的话p16, p21, HP1 gamma以及DAPI染出的heterochromatin foci能取代吗? |
|
|
F*****e 发帖数: 182 | 19 用mouse tissue paraffin section做RNA in situ hybridization,最近反复的摸索条
件,RNA probe的浓度加到很高(5 ug/ml),信号强度才勉强可以接受。我用的10um的
片子,DIG-labeled RNA probe。请问做过的朋友怎样能提高灵敏度?多谢! |
|
h******u 发帖数: 602 | 20 做铁代谢方面的,包括hepcidin, TfR, ferritin and ferroportin。手头的几个抗
体都是做western blot的。现想用paraffin-embedded tissue做免疫组化。大家给
个建议?免疫组化我没啥经验。谢谢拉。 |
|
n***w 发帖数: 2405 | 21 I used some antibodies from abcam to do IHC but I used frozen sections.
I am not sure of paraffin sections. |
|
v*********d 发帖数: 382 | 22 Paraffin 包埋的不基本上都是PFA fix过的吗?
Qiagen的 |
|
p******d 发帖数: 3737 | 23 想看YFP啊。做YFP的paraffin的staining不work啊!!!前两天还上来问来着的。忒悲
催了!!! |
|
|
|
n***w 发帖数: 2405 | 26 或者你可以用点formalin之类的固定,比如说PREFER,PERFIX,等等。
然后送去做paraffin section
如果自己做frozen section, 4%PFA 可以了,没必要加glutaraldehyde. |
|
g***y 发帖数: 201 | 27 CaM Kinase II alpha subunit (AB-1),mouse monoclonal, from calbiochem, cat#
NB12
works on 4%PFA fixed paraffin section, need citrate buffer antigen retrieval
though, dilution 1:200-1:500
also works on crysection.
爱卿免跪平身,千万别忘了给我几个包子! |
|
b*********b 发帖数: 64 | 28 如果你很想了解confocal或者更高级的two-photon,下面两篇paper对你会有帮助。
1.Ojcius DM et al.Immunology and the confocal microscope. Res Immunol 1996
2.Cahalan MD et al.Two-photon tissue imaging: seeing the immune system in a
fresh light.Nat Rev Immunol 2002
ZL1999 (胖头猫)提到了confocal和一般fluoresent microscopy的区别,除了有一点我
不太认同,根据我的理解,confocal microscopy的photobleaching问题应该比一般的
fluoresent microscopy要小,因为前者illuminate smaller volume of your sample。
如果你要做的是培养的细胞,你可以不用改protocal,我一般用8-well chamber slide
,这样一张slide上可以有8个samples。coversl... 阅读全帖 |
|
a*****2 发帖数: 175 | 29 We use GM130 antibody from BD Bioscience that works very well even in
chicken cells. Also I only use 3%BSA for blocking. Paraffin section doesn't
work for Golgi neither based on my experience.
+L |
|
n***w 发帖数: 2405 | 30 起码我用的millipore hamster anti-cd31在paraffin上不好使。你可以试试BD
pharmingen的。
macrophage的话,还可以用CD45. 但是我没有做过染色。 |
|
C**S 发帖数: 522 | 31 BD的CD31抗体在paraffin切片上不好用,只能做冰冻切片。 |
|
y****y 发帖数: 123 | 32 德国一家公司的CD31据说是唯一可以染老鼠paraffin的
但是好像二抗那步比较麻烦
明天去看看号
顺便问一下BD的CD31染冰冻用什么固定比较好?
formalin可以么 |
|
|
g***y 发帖数: 201 | 34 MAP2 from sigma, mouse monoclonal, #M9942
works great on 4% PFA fixed paraffin sections,
also works on cryo-sections, and IF of primary neurons. |
|
g***y 发帖数: 201 | 35 MAP2 from sigma, mouse monoclonal, #M9942
We use it on mouse brain sections all the time.
For paraffin section, it requires antigen retrieval step (microwaving in pH6
.0 10mM citrate buffer).
For IHC on crysections, no AR is required. |
|
D*a 发帖数: 6830 | 36 paraffin切片不就是皱的么,载玻片滴水,放在37度台上,挑切片放到水的表面,泡一
会就开了,把载玻片倾斜,把水倒了 |
|
w*********n 发帖数: 439 | 37 Before doing section, put your paraffin block in -20 for 30min.
When you do sectioning, using wet tissue paper to wet the cutting surface of
your block.
Using warm water to float your tissue section.
Every thing should be fine. |
|
y****y 发帖数: 123 | 38 最好是兔子的
主要做WB和paraffin的IHC
谢谢 |
|
s******r 发帖数: 2876 | 39 能认识mouse IL18,paraffin切片能做IHC或者IF的,
买了两个都不好使。 |
|
P******r 发帖数: 966 | 40 protocol写了fix overnight,但是忘了放在4% PFA有五天之久,还能做staining吗? |
|
y**u 发帖数: 7459 | 41 做什么stain? H&E应该没什么问题,fluorescence有问题 |
|
P******r 发帖数: 966 | 42 fluorescence的自发荧光加强吗?有没有办法去掉影响?sample很重要啊,我真想打我
这个猪脑袋,竟然一忙忘把样品捞出来了。 |
|
y**u 发帖数: 7459 | 43 不是,我记不清具体的了,PFA会quench掉你的antigen,你还是可以试试,可能信号会
很弱。我一般做fluorescence会把样品切小或薄,只fix一两个小时室温 |
|
D*a 发帖数: 6830 | 44 肯定是有影响,如果不想扔就只能切切看了。但是不能跟其他overnight的样品比较信
号强度了。 |
|
a*****t 发帖数: 81 | 45 google antigen retrieval protocol, within one week in pfa still works for
many antigens |
|
P******r 发帖数: 966 | 46 我们的staining标准protocol都有antigen retrieval一步,不过本来那个抗体的信号
就比较微弱,这么一来就怕信号没了。真希望还有得救。 |
|
P******r 发帖数: 966 | 47 control的tissue包埋在一起,问题就怕没有信号或者自发荧光啥的。 |
|
|
D*a 发帖数: 6830 | 49 随便什么温箱都行啊,做paraffin什么的不也要60多度吗 |
|
n***w 发帖数: 2405 | 50 We sometimes fix our mouse tissues with prefer and do paraffin sections, and
after all the organic solvents the signal is gone. |
|