Dear All!

I would like to ask help for a protocol I am currently trying to establish. I am using a protocol (see attached word doc) based on the works of Quinet et al. 2017.

The fibers look like either a mass and didn’t separate nicely, or running nicely parallel, but I can’t see the beginning or ending of the fibers. Sometimes one labeling overshadow the other without any rhyme or reason. I use RPE1 p53-/- cells, which are widely used for this technique. I use the same labor for the experiment, make sure the fibers travel slow enough (15 min) along the slide, and we ordered the same compounds (with the same cat number) others are using. I incubate with CldU for 20 minutes, then with IdU for 1 hour, so there should be short green-long red fibers, but it is either long green or long red, and sometimes yellow, due to overlapping.

I would appreciate any help, insight, thank you!