I use DAPI or Hoechst 33342 to mark nuclei in cells stained by immunofluorescence. I see a red shift in the emission, so the nuclei now appear orange, and are difficult to detect with the usual DAPI/Hoechst filter settings on our confocal. I am using Mowiol mounting medium. I see the same problem with both DAPI and Hoechst on separate occasions. Does anyone know what I can do to prevent this? It happens on cell culture monolayers and on tissue sections.
I am not aware of Hoechst 33342 dye to mark the cell nuclei but I have extensively used DAPI to stain cell nuclei as you could see below in my publications.
Article Localization of epithelial sodium channel (ENaC) and CFTR in...
Article Mapping the sites of localization of epithelial sodium chann...
I have never encountered such problems during my experiments as you stated. However it is quite difficult to resolve the issue just based upon your statement but what I could recommend you to change the mounting medium. I had used the mounting medium that include n-propyl gallate as a anti-fade reagent.
Below is the recipe to make the mounting medium that i used during my experiments:
Buffered glycerol with anti-fade:
10 ml: 0.1 M Phophate buffer (pH 7.4) or 0.1 M Tris-buffer (pH 9.0)
90 ml: Glycerol
500 mg: n-propyl gallate
Perform your experiments with this mounting medium and let me know your results.
did you see this red shift in a merged image, or did you use other antibodies that acquire red color ?
Thanks Sachin Sharma for the recipe. I will try it out.
Ayman Khalifa thanks for your input - the red shift is visible when scanning just for the DAPI. I have sometimes used antibodies tagged with Alexa 488 and 568 at the same time, but I see the shift when the antibodies are not present too. In addition I scan the samples with a sequential scan so would not expect any crosstalk between the DAPI and antibodies when they are present.
this paper might be informative: Article Photoconversion of DAPI and Hoechst dyes to green and red-em...