I would like to determine whether dormant cells in situ/in vivo on tissue sections have lower RNA content. In flow cytometry pyronin Y and Hoechst or DAPI seem to be sometimes used for this purpose.
I have never done such stains and I am not sure whether mRNA/ribosomal RNA can really be stained on a tissue section with pyronin Y after blocking nuclear staining with DAPi or Hoechst.
Has anybody tried to identify "G0 cells" or dormant stem cells in situ this way? The publications I found do not use the fluorescent side of pyronin Y and I frankly cannot identify anything on the figures in these publications. There is this idea out there in flow cytometry that G0 cells have low RNA content, probably due to the fact or the reasoning of the investigators, that dormant/G0 cells don't do much and do not intend to do much in the near future and therefore do not need RNA (neither ribosomal, nor mRNA). Basically it seems to be a ribosomal RNA stain, doesn't it?!
Bottom line: I would like to stain the RNA in the cytoplasm after staining the DNA in the nucleus and determine a rough ration between the two on tissue sections. Any ideas comments?
Key words: fluorescent dyes, in situ staining, frozen or FFPE tissue sections, G0 phase, dormant stem cells, pyronin Y.
Dear Professor Thomas, i was also curious about identifying the G0 stage of my cells. So, I was looking for reagents relevant to G0 cells. Someone from my school is using Pyronin Y along with 7-AAD in flow cytometry to identify G0 phase but its on cultured cells. However, I found that some companies are selling `Methyl Green Pyronin` to stain RNA (cytoplasm) and DNA (nuclei) in FFPE tissue sections. I haven`t used it by myself yet. I just considered Ki-67 negative and label retained cells as dormant. Do you think Methyl Green Pyronin is also applicable to G0/dormant cells?
Thanks Ruhul! I am looking for something for a fluorescent application. I think I will just try the pyronin Y and the Hoechst dye and see how it looks in my microscope. Since the vast majority of RNA is ribosomal, a ribosomal protein stain may do the same trick??
According to a recent paper (Coller et al. 2006. Plos Biol.) and based on my expression data, I could observe distinct gene expression programs in response to different signal. If ribosomal proteins are also regulated in the similar fashion, I think it should work. Best wishes,
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