【在 b*****l 的大作中提到】 : 没看懂。。。是说同一套 kit,镜下没问题,FACS 有问题?两者的 denature 方法一 : 样么?还是用的不同的 antibody/方法?另外,镜下和 FACS 可以交叉验证的:FACS : 染色后可以 mount 到片子上镜下看,而为镜下染色用的 kit 也可以尝试做 FACS 来对 : 照。还有,这个 brdu-kit 在你手下曾经 work 过么?如果第一次用,是不是先找个类 : 似的 cell line 做个 in vitro 的 positive control 先?
m*p
14 楼
That makes sense a lot! In P0-P7 hearts, very few cells proliferate, and after P7, there is almost no cell proliferation, unless you damage the heart. However, when you isolate cells in the culture, the situation will be different as more cells proliferate.
【在 m*p 的大作中提到】 : That makes sense a lot! In P0-P7 hearts, very few cells proliferate, and : after P7, there is almost no cell : proliferation, unless you damage the heart. However, when you isolate cells : in the culture, the situation will : be different as more cells proliferate. : : BrDu
m*p
16 楼
They did not say how long they pulse label the heart with BrdU. But, since their data indicates the percentage of BrdU positive cells is almost equal to Ki67 percentage, that means the BrdU labeling time will be more than 24 hours. When you label an E14.5 hearts with Brdu for 24 hours, more than half of the cardiomyoctyes will be positive if you know how fast the embryonic cells proliferate in vivo, however they only found about 20% cells were positive. Their Ki67 staining is not the right staining, if you know what cell cycle phases will be Ki67 positive. Some of the cells they point to are not cardiomyocytes. BrdU staining is horrible. I do not know what I can tell you, but since you can get the IHC work, then try IHC and count the percentage of BrdU positive cells. Also, Ki63 and pH3 only give you a snap shop information, while BrdU labeling is a accumulation of proliferative cells after you inject the BrdU, so the time you pulse label the heart determines Brdu percentage.
是啊是啊。建议 lz 做一个 in vivo positive control,也就是说,用一种一定能够 使心肌细胞 prolif 的方法处理一下,比如说人为造成心肌损伤,然后做 FACS 看看是 否能看到 brdu 信号。
cells
【在 m*p 的大作中提到】 : That makes sense a lot! In P0-P7 hearts, very few cells proliferate, and : after P7, there is almost no cell : proliferation, unless you damage the heart. However, when you isolate cells : in the culture, the situation will : be different as more cells proliferate. : : BrDu
M*n
18 楼
try EdU. a bit more expensive, but easier to handle.