Possibly switching methods: To section or not to section?
posted by: acarr
John G. and I have been working on a protocol to fluorescently label zebrafish embryos using the wholemount approach. But labeling the entire embryo has its challenges: there is background non-specific staining from the secondary antibody (color), and the positive signal is not strong (i.e., you can see labeled cells, but the cells are faint in color). Also it is difficult to take a good, sharp/clear picture on the microscope….everything seems blurry.
So, yesterday I cross-sectioned some embryos, and put the sections on microscope slides. And, today John started to label those cross sections using the same antibodies. I also sectioned some mutants and will add them to the labeling project today. We will see tomorrow if the sectioning method provides a clearer image versus the whole-mount staining approach.
To tell you the truth, I thought the whole-mount labeling method would be the easier approach, and provide the best image of all the TH-positive cells. There is only a small number of these cells, so sectioning could mean seeing about 3-8 cells in each section, whereas in the whole-mount you could see 20+ cells at one time…..but if I get better results by sectioning, I will spend the time doing so.