Hello,

I have been using EdUClickIT kit on P2 neonatal whole mount mouse pancreata. Sometimes it works and I detect EdU+ cells, sometimes it doesn't work (even for a pancreas from the same litter that worked for another pancreas!). Any suggestions of what I might be doing wrong? Which factors might play a role? PFA fixation length? ClickIT reaction mix incubation duration? Any other suggestions?

Generally the protocol I follow after collection of tissue is:

1. PFA fixation overnight

2. PBS washes

3. Incubation in 0.5%Triton100X, 2%serum in PBS - I have tried variable length from 4h to overnight

4. Incubation in EdU ClickIT reaction mix from Invitrogen - I have tried variable lengths from 4h at room T to overnight at 4C.

5. Washes in 2% donkey serum 3 times, at least 10 minutes.

6. Washes in PBS, 3 times, 30 minutes

Any suggestions on how my protocol could be refined would be highly appreciated.

Many thanks,

Magda