Hello Everyone,
Does anyone have problems with PI during cell staining for microscopic imaging of mammalian cells?
After fixation of cells on cover glasses and incubation with my nanoparticles, to stain the nucleus, I am using PI (because of the blue fluorescent emission of my particles -- I couldnt use DAPI or Hoescht directly--) as 1:3000 dilution (from 1mg/mL stock solution in PBS) in PBS. And I am incubating cells with 600 uL PI for 10 min in room temperature. After incubations, I am washing the cover glasses with 1:30 methanol:water solution as three times. However, i could just catch nice staning with a spherical nucleus structure (like in DAPI) once. What am I doing wrong? In my images, PI is also distrubuted to cytoplasm, somehow.
Second question is about other filters. For PI, I m using TX Red filter but is there any explanation with why i see PI in blue and green emission region as well ?
Have a nice week!
Hello Cristina,
As you guessed, i did the fixation steps by firstly fixing cells with 3% PFA and washing with Glycine buffer and incubation with particles (again as you guessed for attaching them to cell surface) and PI. And I m thankful to you for explaining this nicely. As you said, i also saw today, it depends on cells :) En fin, thanks for giving me other oppurtinities for nuclei staining.
Best wishes to you as well,
Bilal
Last paragraph of what Christina told seems to be the problem. PI can not penetrate cell membranes in healthy cells (That's why we use permeabilization in cell cycle studies and do not use any permeabilization step when used in conjuction with Annexin V to determine apoptosis/necrosis).
Roughly you could add perm. step before PI : fixation/NP treatment/permeabilization/PI treatment
However, I would try NP treatment/fixation/permeabilization/PI if possible.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining