Sc 13091

Organoids were retrieved from Matrigel using Cell Recovery Solution (Corning) and fixed in 4% paraformaldehyde in PBS (Alfa Aesar) for 30 min at room temperature. 2D cultures on glass coverslips were fixed in 4% paraformaldehyde in PBS for 20 min. Immunostaining was performed as previously described [17] . Rabbit polyclonal antibodies against proglucagon (Santa Cruz, sc-13091) were used at 1:200 and goat polyclonal antibodies against GFP (Abcam, ab5450) at 1:1000 to detect YFP and GCaMP3. Secondary antibodies conjugated to Alexa-Fluor 488 and 555 (Invitrogen) were used at 1:1000 and Hoescht (Sigma) nuclear stain at 1:3000.

Primary antibodies used were: guinea-pig anti-insulin (ab7842, 1:200; Abcam), rabbit anti-glucagon (SC13091, 1:200; SantaCruz, Santa Cruz, CA, USA). Sections were counterstained using Hoechst (1:1300, Invitrogen, UK). Stained sections were imaged using either the incubated Zeiss LSM510 META Confocal Microscope, Zeiss LSM710 META Confocal Microscope, Zeiss AxioImager Motorised Upright Microscope. Images were analysed using Zen software.

Mouse pancreata were extracted and fixed in 10% (v/v) neutral buffered formalin at 4 °C for 18 h before paraffin embedding and sectioning. Sections were labelled with polyclonal anti-insulin (Dako A4056, 1:200 dilution, Secondary-Alexa 488 1:1,000) or anti-glucagon (Sigma, G2654 or Santa Cruz, sc-13091, 1:200 dilution, Secondary-Alexa 568 1:500) antibodies and sealed using Vectashield hardset mounting medium with DAPI (Vector Laboratories). Fluorophores were excited using a Zeiss Observer Vivatome and a 20x/NA 0.8 x objective and 365 nm, 470-nm and 540–580-nm LEDs. Emission was detected using Semrock filters centred on 377/50 nm, 472/30 nm, and 534/20 nm for DAPI, Alexa 488, and Alexa 568, respectively.