f.carlotti@lumc.nl
26 november 2020 bij 18:05- Antwoorden
Nice presentation! I have 2 questions for you 🙂
GLP1-Receptor antibodies are notoriously difficult: how did you validate the specificity of the antibody you used?
And can you use your labelled-Exendin4 on sections of T1D ? that would be interesting to compare to the GLP1-R staining.
M. Boss (Nijmegen)
26 november 2020 bij 18:16- Antwoorden
Thank you! We have had problems with GLP-1R antibodies in the past as well. The antibody we used now is: GLP-R1, Mab 3F52 2.5 ug/ml, DSHB, RRID: AB_2618100, which seems to work well. It binds to the external part of the receptor and its binding can be blocked with GLP-1. We are still in the process of further examining these slides with other markers, for example for beta cells, to check our results.
We could use radiolabeled exendin for in vitro autoradiography in theory, although this has never worked very well in our hands. We do also have different fluorescently labeled compounds which could be a possibility. The problem here is the low signal. When using radiolabeled exendin in vivo, you get high uptake in the beta cells because of metabolic trapping. This enables us to do in vivo as well as ex vivo imaging. With in vitro work the signal is much lower.
f.carlotti@lumc.nl
26 november 2020 bij 19:08- Antwoorden
ok, thank you for your detailed answer and all the best with the continuation of your project
3 Reacties
Nice presentation! I have 2 questions for you 🙂
GLP1-Receptor antibodies are notoriously difficult: how did you validate the specificity of the antibody you used?
And can you use your labelled-Exendin4 on sections of T1D ? that would be interesting to compare to the GLP1-R staining.
Thank you! We have had problems with GLP-1R antibodies in the past as well. The antibody we used now is: GLP-R1, Mab 3F52 2.5 ug/ml, DSHB, RRID: AB_2618100, which seems to work well. It binds to the external part of the receptor and its binding can be blocked with GLP-1. We are still in the process of further examining these slides with other markers, for example for beta cells, to check our results.
We could use radiolabeled exendin for in vitro autoradiography in theory, although this has never worked very well in our hands. We do also have different fluorescently labeled compounds which could be a possibility. The problem here is the low signal. When using radiolabeled exendin in vivo, you get high uptake in the beta cells because of metabolic trapping. This enables us to do in vivo as well as ex vivo imaging. With in vitro work the signal is much lower.
ok, thank you for your detailed answer and all the best with the continuation of your project