Goat anti rat igg h l

Immunofluorescence experiment and analysis of Glu-Cre⁺; GCaMP6f^f/+,and Ins2-RCaMP1.07 mice was performed as previously described^{75 ,76} . Islets were fixed in 4% Paraformaldehyde Fix Solution for 1 h, and were blocked overnight in PBS, and 0.3% Triton-100X at room temperature. The samples were incubated overnight days at 4 °C in a guinea pig anti-insulin antibody (1:200, A0564, Dako), a rat anti-somatostatin antibody (1:200, ab30788, Abcam), and a mouse anti-glucagon antibody (1:200, G2654, Sigma) separately. The islets were then incubated for 2 h at 4 °C separately with Goat anti-Guinea pig immunoglobulin G (IgG) (H+L) secondary antibody (DyLight^TM 488) (1:1000, SA5-10094, Invitrogen), Goat anti-Guinea pig IgG H&L (DyLight^TM 550) (1:1000, SA5-10095, Invitrogen), Goat anti-Rat IgG H&L (Alexa Fluor 568) (1:1000, A-11077, Invitrogen), Goat anti-Rat IgG H&L (Alexa Fluor 350) (1:1000, A-21093, Invitrogen), Goat anti-Mouse IgG H&L (Alexa Fluor 488) (1:1000, A32766, Invitrogen), and Goat anti-Mouse IgG H&L Cy3 (1:1000, A10521, Molecular Probes).

Tumors were collected from the mice and snap-frozen in optimal cutting temperature medium. Tumor sections were cut using a cryotome, mounted on slides and stained with different primary antibodies, including CD8 (Abcam, cat. no. ab22378) and CD80 (Abcam, cat. no. ab100790), overnight at 4 °C following the manufacturer’s instructions. Following the addition of fluorescently labeled secondary antibodies (goat anti-rat IgG (H + L; Thermo Fisher Scientific, cat. no. A18866) and goat anti-rabbit IgG (H + L; Thermo Fisher Scientific, Cat. no. A32733)), the slides were analyzed with high-speed confocal microscopy (Andor Dragonfly 200, UK). All antibodies used in the experiments were diluted 200 times.

For BrdU immunodetection, antigen retrieval was performed before blocking: the samples were kept overnight in Tris-buffer (pH 9) at 65°C and then washed with PBS for 15 min and incubated with 2N HCl for 2 h at 37°C, followed by 10-min neutralization in 0.1 M borate buffer (pH 8.5). Then slices were incubated in 0.1% Tween 20, 0.3 M glycine, and 1% BSA in PBS for 6 h. After blocking, samples were incubated overnight with an anti-BrdU antibody (1:250; Abcam). Non-bound antibodies were washed five times in PBS/1% FBS/0.1% Tween-20. Samples were treated for 2 h at room temperature with Cy3-conjugated secondary polyclonal antibodies (goat anti–rat IgG H + L, 1:750; Thermo Fisher Scientific). Nuclei were counterstained with Hoechst 33342. Images were captured on an Eclipse Ti-E microscope with the A1 confocal module (Nikon Corporation) and a Plan Fluor 40×1.3 objective.