Goat anti rabbit igg conjugated to alexa fluor 488

In the case of paraffin embedded tissues a standard rehydration protocol for 6 μm sections was applied as described earlier (Scholven et al., 2009 (link)). To reduce auto-fluorescence, sections were immersed in 0.1% Sudan Black diluted in 70% ethanol for 20 min at RT, followed by washing 5 min in 50% ethanol and rehydration in PBS (PBS including 1% BSA) 10 min at RT. Antigen retrieval was achieved by boiling in 10 mM citrate buffer, pH 6, for 40 min. Non-specific binding of the primary antibody was blocked by incubation in 10% goat serum in PBS (1% BSA in PBS) for 1 h at RT. Mucous and cell membranes were stained using an Alexa Fluor 594 conjugate of wheat germ agglutinin (Life Technologies) at a concentration of 5.0 μg/mL HBSS for 15 min at RT, followed by washing for 20 min in PBS. Rabbit monoclonal anti-NR1D1 antibody (Abcam, EPR10376, 0.086 mg/ml) was used at a 1:50 dilution (PBS with 1% BSA). A goat anti-rabbit IgG conjugated to Alexa Fluor 488 (pre-absorbed, Abcam) was used as the secondary antibody at a dilution of 1:400 for 1 h at RT. Incubations without the primary antibody, or with rabbit IgG1, were performed as negative controls. Nuclei were counterstained using 200 ng/ml DAPI (Roche) for 10 min, and sections were embedded in 50% glycerol in PBS.

Recombinant human insulin was purchased from Sigma Aldrich (St. Louis, MO, USA). Antibiotic-antimitotic solution, trypsin–EDTA solution 0.25%, Hank’s Balanced Salt Solution (HBSS) without phenol red, Dulbecco’s Modified Eagle’s Media (DMEM) with glutamine, Fetal Bovine Serum (FBS), Hoechst 33258, Pentahydrate (bis-Benzimide)-FluoroPure, MitoTracker Red FM (MTR) and methanol-free formaldehyde (16% solution) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Methanol, Acetonitrile, and water were Optim LC–MS Grade and obtained from Fisher Scientific (Hampton, NH, USA). Ethanol LiChrosolv Grade was obtained from Merck KGaA (Darmstadt, Germany). Rabbit anti-Von Willebrand factor (vWf) antibody and goat anti-rabbit IgG conjugated to Alexa Fluor 488 were obtained from Abcam (Cambridge, MA, USA).