Goat anti guinea pig igg

Pancreatic tissue was collected from six wildtype 129/S6-SvEv-TC1 mice and fixed in 4% PFA at 4°C overnight. The tissue was removed from PFA and washed in PBS before dehydrating and embedding in paraffin. Paraffin sections were cut to 5 µm thickness. RNAscope in situ hybridisation to detect RNA transcripts was performed according to the manufacturer protocol (Advanced Cell Diagnostics) using probes for RAMP1 (#532681) and RAMP3 (#497131). Antibody staining was performed following in situ hybridisation with guinea pig anti-insulin antibody (Invitrogen, diluted 1:1000) and rabbit anti-glucagon antibody (ZYMED, diluted 1:300) in 3% BSA in PBS with 0.1% Triton X overnight at 4°C. This was followed by secondary antibody staining with goat anti-guinea pig IgG (Jackson ImmunoResearch, diluted 1:400) and donkey anti-rabbit IgG (Jackson ImmunoResearch, diluted 1:200), respectively, for 1 hour at room temperature.

The following primary antibodies were used: Guinea pig anti-Tango1 (1:1,000) (Lerner et al., 2013) , rabbit anti-GM130 (1:500, Abcam, cat # ab30637) and goat anti-Gmap (1:500) (Riedel et al., 2016) . Secondary antibodies were Goat Anti-Guinea Pig IgG (Rhodamine conjugated, Jackson ImmunoResearch, cat # 106025003; Alexa Fluor 647 conjugated, Jackson ImmunoResearch, cat # 106605003), Alexa Fluor 555 Donkey Anti-Rabbit IgG (Invitrogen, cat # 1945911) and Alexa Fluor 555 Donkey Anti-Goat IgG (Abcam, cat # ab150130) respectively. Antibody stainings were perfomed using standard procedures for larval tissues. Briefly, larvae were predissected in PBS, fixed in PBS containing 4% PFA (paraformaldehyde, Sinopharm Chemical Reagent, cat # 80096692), washed in PBS (3 × 10 min), blocked in PBT-BSA [PBS containing 0.1% Triton X-100 detergent (Sigma-Aldrich, cat # T8787), 1% BSA (Zhongkekeao, cat # 201903A28), and 250 mM NaCl (Amresco, cat # 0805C384)], incubated overnight with primary antibody in PBT-BSA in 4°C, washed in PBT-BSA (3 × 20 min), incubated for 2 h with secondary antibody in PBT-BSA at RT, washed in PBT-BSA (3 × 20 min) and PBS (3 × 10 min). Tissues were finally dissected and mounted on a glass slide with a DAPI-Vectashield (Vector Laboratories, cat # H-1200).