Goat anti guinea pig igg alexa fluor 555

Brainstems of mice were collected after 4% PFA perfusion and post-fixed for 4 hours on ice. After cryoprotected in 30% (w/v) sucrose for two nights at 4 °C, brainstem was immerged into OCT and sectioned at 50 μm using the cryostat for free-floating section staining. Sectioned tissues were blocked with 10% goat serum in PBST (PBS containing 0.1% Triton X-100) for 1 hour and incubated with primary antibodies at 4 °C overnight. After washing with PBST, secondary antibodies were applied for 2 hours at room temperature. Fluorescence images were taken using Nikon C2 confocal system. Primary antibodies including: rabbit anti-GFP (Invitrogen, A11122, Lot# 1925070; 1:1000), rabbit anti-c-Fos (Santa Cruz Biotechnology, sc-52, Lot# B0112; 1:1000), mouse anti-NeuN (MilliporeSigma, MAB377, Lot#3205920; 1:1000), goat anti-WGA (Vector Laboratories, AS-2024, Lot#T1112; 1:1000), guinea pig anti-Synaptophysin 1 (Synaptic System, 101 004; 1:200). Secondary antibodies including: goat anti-rabbit IgG-Alexa Fluor-488 (Invitrogen, A11008, Lot # 1797971; 1:500), goat anti-rabbit IgG-Alexa Fluor-555 (Invitrogen, A21429, Lot # 1683674; 1:500), Goat anti-mouse IgG-Alexa Fluor-Cyanine5 (Invitrogen, A10524, 1:500), Cy™3 AffiniPure Donkey Anti-Goat IgG (Jackson ImmunoResearch, 705165147, Lot # 148575; 1:500), goat anti-guinea pig IgG-Alexa Fluor-555 (Invitrogen, A21435; 1:500).

Lipofectamine 2000 and 4–12% NuPage gel were purchased from Invitrogen (Carlsbad, CA, USA). Protein phosphatase inhibitor was purchased from Beyotime Biotechnology (Beijing, China). Protein A-Agarose was from Santa Cruz Biotechnology (Dallas, TX, USA). Guinea pig anti-insulin (dilution: 1:2000) was from Merck Millipore (Billerica, MA, USA) and mouse anti-proinsulin antibody was from Novus Biologicals (Littleton, CO, USA). Rabbit anti-Hsp90 (dilution: 1:2000) antibody was from Assay designs (Ann Arbor, MI, USA). Rabbit anti-cleaved caspase 3 antibody was from Cell Signalling Technology (Danvers, MA, USA). Annexin V with Alexa Fluor™ 555 conjugation, Goat anti-guinea pig IgG Alexa Fluor 555 and goat anti-rabbit IgG Alexa Fluor 435 was bought from Invitrogen (Carlsbad, CA, USA). Rabbit anti-Myc antibody was from Immunology Consultants Labs. Horseradish peroxidase–conjugated antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Enhanced chemiluminescence Western blotting substrate was from Millipore (Billerica, MA, USA). Trans³⁵S label and pure ³⁵S-methionine were from PerkinElmer (Waltham, MA, USA).