b******s
发帖数: 1089 | 1 Sorry for English. Can't input chinese in the lab.
Nowadays, people choose to fix samples by high pressure freezing and freeze
substitution more and more. There is no question that this method is elegant
to keep the cellular structure and keeping the antigen active for immuno-EM
.
I read many discussions in EM mail group talking about their papers got
rejected because they used the conventional chemical fixations. It seems
people in EM field have different opinions about requirement of using high
pressure freezing and freeze substitution. the problem is the facility is
super expensive if you want to use this method and no availability in most
cases.
My question is do you know if high class journals nowadays allow the
conventional chemical fixations? I mean if you use this way to do the immuno
-EM and it would be one of the major tools in the paper?
Thanks a lot! |
C*******h
发帖数: 75 | 2 I would say it is very easy to get rejected by glossary journals with the
mentioned-above weakness in your methodology. Isn't possible to colloborate
with someone else? Our institute has this facility. |
s******y
发帖数: 28562 | 3 我不是业内行家所以只能说来参考吧。其实两种方法都有好和不好的地方。
高压冷冻对于保存细微结构比较好，抗染也很漂亮。但是技术不太容易掌握，
弄不好的话就把微结构全部破坏了。而且很多地方没有那个仪器。
化学固定其实没有他们说的那么糟糕，其实大部分结构保存得还是很好的，
但是最大的问题就是做抗染的时候信号／噪音比太差，除非是阳性结果非常
强的情况下不好下结论。
你问的这个问题，关键在于你的实验到底是在做什么，如果是证明某个蛋白在
某个已知的结构上的位置，比方说要证明某蛋白在线粒体的外膜上，那就没有
什么大问题。尤其假如你已经有其他的证据，比方说生化证据和光学成像证据，
那么基本上不会被挑战。
但如果你要做一个大家都不是很了解的结构，那就容易被人拍砖。比方说你要
做一个以前大家都不知道的微管蛋白的聚合中心，而且要有该形态研究作一个大
的结论，那么就容易死得很惨。其实要做形态学发到高端杂志的话，一般会被
要求做两种以上的方法。除非你老板是大牛。
freeze
elegant
EM
【在 b******s 的大作中提到】
: Sorry for English. Can't input chinese in the lab.
: Nowadays, people choose to fix samples by high pressure freezing and freeze
: substitution more and more. There is no question that this method is elegant
: to keep the cellular structure and keeping the antigen active for immuno-EM
: .
: I read many discussions in EM mail group talking about their papers got
: rejected because they used the conventional chemical fixations. It seems
: people in EM field have different opinions about requirement of using high
: pressure freezing and freeze substitution. the problem is the facility is
: super expensive if you want to use this method and no availability in most
|
C*******h
发帖数: 75 | 4 Yours more informative. Nice post!
" I mean if you use this way to do the immuno
-EM and it would be one of the major tools in the paper? " in his/her post.
【在 s******y 的大作中提到】
: 我不是业内行家所以只能说来参考吧。其实两种方法都有好和不好的地方。
: 高压冷冻对于保存细微结构比较好，抗染也很漂亮。但是技术不太容易掌握，
: 弄不好的话就把微结构全部破坏了。而且很多地方没有那个仪器。
: 化学固定其实没有他们说的那么糟糕，其实大部分结构保存得还是很好的，
: 但是最大的问题就是做抗染的时候信号／噪音比太差，除非是阳性结果非常
: 强的情况下不好下结论。
: 你问的这个问题，关键在于你的实验到底是在做什么，如果是证明某个蛋白在
: 某个已知的结构上的位置，比方说要证明某蛋白在线粒体的外膜上，那就没有
: 什么大问题。尤其假如你已经有其他的证据，比方说生化证据和光学成像证据，
: 那么基本上不会被挑战。
|
b******s
发帖数: 1089 | 5 Thanks for the reply. I am also thinking of collaboration if HPF turns out
to be necessary. But I am still in the period of confirming preliminary data
. I showed the images to an EM expert before but she liked the idea of HPF.
Could you let me know where is your institute?
colloborate
【在 C*******h 的大作中提到】
: I would say it is very easy to get rejected by glossary journals with the
: mentioned-above weakness in your methodology. Isn't possible to colloborate
: with someone else? Our institute has this facility.
|
b******s
发帖数: 1089 | 6 Thanks! Good explaination and I found it is similar to many other EM experts
' opinion. Actually HPF also causes artifacts and it won't be able to fix
well large samples.
My work is to find out what pathways the protein use to be secreted. One of
tools I am using now is to use immunoEM to localize proteins to sub-cellular
structures. I know the combination of multiple evidence would be important
to convince people. I will try to homogenize tissues and do the western. But
according to my PI, western is hard for my protein. A student tried this
before my coming but failed. To be frank, I don't trust that student very
much. I would like to give a try myself.
At the other end, however, immuno-EM would be one of my major evidence. I am
just worrying that it won't be accepted by high class journal. You know, i
spent much much time in optimizing the immuno-EM protocols.....
【在 s******y 的大作中提到】
: 我不是业内行家所以只能说来参考吧。其实两种方法都有好和不好的地方。
: 高压冷冻对于保存细微结构比较好，抗染也很漂亮。但是技术不太容易掌握，
: 弄不好的话就把微结构全部破坏了。而且很多地方没有那个仪器。
: 化学固定其实没有他们说的那么糟糕，其实大部分结构保存得还是很好的，
: 但是最大的问题就是做抗染的时候信号／噪音比太差，除非是阳性结果非常
: 强的情况下不好下结论。
: 你问的这个问题，关键在于你的实验到底是在做什么，如果是证明某个蛋白在
: 某个已知的结构上的位置，比方说要证明某蛋白在线粒体的外膜上，那就没有
: 什么大问题。尤其假如你已经有其他的证据，比方说生化证据和光学成像证据，
: 那么基本上不会被挑战。
|
s******y
发帖数: 28562 | 7 在这种情况下，我会主张你先把生化证据收集了再说。如果native protein WB
实在做不出来的话可以考虑用tagged protein.
化学固定法目前还是一个比较通用的方法，速冻电镜方式只对于形态学有这个要求，一
般的生化细胞实验不需要。
对于你说的这个实验，我觉得不需要。我建议你先收集其他证据，等文章完善了，
送出去，万一reviewer 一定要求你做速冻电镜你再做。
experts
of
cellular
important
But
【在 b******s 的大作中提到】
: Thanks! Good explaination and I found it is similar to many other EM experts
: ' opinion. Actually HPF also causes artifacts and it won't be able to fix
: well large samples.
: My work is to find out what pathways the protein use to be secreted. One of
: tools I am using now is to use immunoEM to localize proteins to sub-cellular
: structures. I know the combination of multiple evidence would be important
: to convince people. I will try to homogenize tissues and do the western. But
: according to my PI, western is hard for my protein. A student tried this
: before my coming but failed. To be frank, I don't trust that student very
: much. I would like to give a try myself.
|
