Hi Abu Saleh,

If your mouse anti-glucagon and mouse anti-Ki67 are of different isotypes you can still use them together having secondary, isotype specific antibodies. If both antibodies are of the same isotype you could label them directly with two different fluorophores. Many easy commercial kits are now available to do that.

Good luck!

Hi Moin, 

I am guessing Ab might not be the problem unless they were badly handled or exposed to bright lights for a long time prior to use.  Since you feel that Goat anti-glucagon worked fine but the signal faded away, I am guessing that it could be due to over exposure to laser beams for a long time or the laser beam itself was used at a high laser power if you were using a confocal microscope (CM). Photobleaching is a common problem that can be fixed. Minimum exposure to laser might be the solution to avoid bleaching of your fluorophore. If you are using a CM, try increasing the scan speed and start with low laser power, which is minimum enough to see the signal and position your sample for imaging. This way you will make sure the laser beam doesn't bleach your fluorophore. Once you have the sample in focus, use your desired laser scan speed and laser power to image your sample. Even if you expose your samples to low power laser for a long time, you might still see bleaching. Likewise, if you increase scan speed and still expose your samples to high laser power, the effect could still be bleaching. So you might want to be cautious there. I hope this helps. 

Best, 

Gnaneshwar

Hi Yadav, Thanks for your comment. You pointed out the most primary reason of photobleaching (over exposure to light). But this option was taken into consideration and it was confirmed that exposure to light was not the issue. The staining disappeared even within a minute !! whereas I can still image the slides stained with mouse anti glucagon Ab even after 3 months!! And the experiment was performed independently for multiple times. However, good news is I have solved the problem.

Thanks to Dr. Anna Albecka ! Based on her idea I discussed with tech support of Jackson Immuno Research Lab. They recommended a strategy of using monovalent Fab fragments to distinguish between the two mouse primaries. You can find some examples in the following link. Based on your primary Ab, host and secondary Ab you need to select the appropriate combination of Fab fragments. it is recommended to select Fab which is pre-absorbed in multiple species.

https://www.jacksonimmuno.com/technical/products/protocols/double-labeling-same-species-primary

Thanks again for your response.