Propidium iodide staining of nucleus for IFA - troubleshooting required (Jun/25/2008 )
I've used propidium iodide to counterstain the nucleus and what I've got under the microscope was red fluorescence all over the cell. I'm not using rnase but had permeabilized the cell with triton x-100. Some protocol mentioned using rnase while others didn't. Anyone did counterstain using propidium iodide? Is is crucial to treat cells with rnase prior to staining with PI? Thanks for your help.
P.s. I'm not using a blue fluorescence dye because i'd like to look at green with red and blue seems to lack the 'contrast'. Furthermore, it requires UV laser which I'm not sure is working or not for the time being...
In my experience, RNase treatment can be really useful but is not always required depending on your other steps in immunofluorescence.
If you do need it (i.e lots of cytoplasm in your cells) I have previously found that 1 hour treatment with RNase A 2mg/ml in PBS at 37C was good, followed by DNA counterstaining with 25ug/ml propidium iodide in dH2O at room temp for 30 minutes in the dark. BUT for other cells just a quick 5 minutes in 50ug/ml PI was really good and stained only the nucleus.
Two of the most important things I found were to prepare the PI yourself from powder - make a 1mg/ml stock and store at -20 in the dark, and to make it with water and not PBS - I don't know why it helped, but it did
I'll give it a try.
I'll give it a try.
I recently started doing TUNEL with using IHC and also use PI for a counterstain. I do not treat with rnase and still seem to get pretty good staining of practically all my cells.
I've tried staining the cells again, this time with Rnase before PI and the result showed lower cytoplasmic noise. Previously, tried without Rnase and sometimes there's low and sometimes there's high background noise from RNA. I'm guessing it's the permeabilization steps that might cause the inconsistency, but using RNase seems to make sure the cytoplasmic background remains low. Hey, green and red seems to be like a great combination of color. It's striking compared to blue and green. Haha... but if there's overlap of color, it's somewhat yellow, compared to cyan for blue and green.
I was wondering if you cultured your cells on the slides or attached them after a certain point? I am trying to do everything in tubes and then attach the cells to slides but do not know how to do this exactly. Any advice would be appreciated. And I agree, green and red is very nice, lol.
I cultured my cells on coverslip (sometimes coated with poly d lysine if the cells are less adherent) and work from there. I've not tried staining it while in suspension, after which immobilizing it onto slides. But I've encountered the method while googling. Most used cytospin while some prefered blood smearing method. Previously I've tried blood smearing, the quality was ok, but the cells were sparse (Note: that was when I was working on trypan blue exclusion, not fluorescence staining). Best wishes.