Abstract
This protocol aims to introduce methods for immunostaining two endogenous proteins insulin and glucagon and one exogenous transgene driven EGFP in mouse pancreatic islet. The immunostaining results of insulin and glucagon indirectly tell functionality of pancreatic beta cells and alpha cells respectively. Furthermore, the protocol provides immunostaining steps for the third protein which can be applicable to any other endogenous proteins with a specific antibody generated in mouse.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Triple immunostaining for EGFR, insulin, and glucagon in a pancreatic tissue section. Pancreatic tissue sections were obtained from mice 3 weeks after Tam administration to induce EGFP from a transgene. Pancreatic tissue sections were triple immunostained for EGFP (Green), insulin (Red), and glucagon (Blue), and representative single-channel fluorescence images are shown individually and merged (lower-right image of each group). [Please cite Reference 1 (Figure 1B)]
Recipes
Acknowledgments
This work was supported in part by the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology Grants 2011-0011433, 2012M3A9C3048686 and 2014R1A1A4A01004329 (S.H.B) and NIH grants DK42394, HL52173, and HL057346 (R.J.K.). R.J.K. was an Investigator of the Howard Hughes Medical Institute. This protocol was adapted from Back et al. (2009), and a first short version of the adapted protocol was published in Han et al. (2013).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.