A10521

Triple immunofluorescent staining of LDLR, InsR, and p-AMPK in hepG2 cells and liver tissue, was conducted by incubating simultaneously with a mouse InsR monoclonal antibody (1:50, sc-57342, Santa Cruz, USA), rabbit polyclonal antibody against p(Thr183/172)-AMPKα1/2 (1:100, YP0575, ImmunoWay, USA) and Alexa Fluor 488-conjugated rabbit LDLR antibody (1:100, ab196377, Abcam, UK). Then, samples were stained with CY3-conjugated goat anti-mouse secondary antibody (1:200, A10521, Thermofisher, USA) and Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (1:200, A32728, Thermofisher, USA). For double staining of IL-6 and TNF-α in liver and epididymal fat tissues, samples were stained with rabbit IL-6 (1:100, GB11117, Servicebio, China) and mouse TNF-α (1:100, GB11188, Servicebio, China) primary antibody and then incubated with FITC-conjugated goat anti-mouse secondary antibody (1:100, A16079, Thermo Fisher, USA) and CY3-conjugated goat anti-rabbit secondary antibody (1:100, A10520, Thermo Fisher, USA). Finally, all samples were mounted with DAPI. Fluorescence pictures were photographed on a C2t Nikon fluorescent microscope. The excitation at 377 nm was for nucleus, 494 nm for LDLR and TNF-α, 543 nm for InsR and IL-6, 628 nm for p-AMPK. The emission was collected at 447 nm for nucleus, 527 nm for LDLR and TNF-α, 586 nm for InsR and IL-6, 690 nm for p-AMPK.

Cells were grown in monolayer on coverslips for 48 h +/− 10 μM Trimethoprim (TMP). Medium was removed and cells were fixed and permeablised with 4% formaldehyde and 0.1% Triton for 20 min at room temperature. Non-specific binding was blocked using 1% BSA before antibody staining: rabbit anti-GFP (ab290, Abcam), Phalloidin-TRITC (P1951, SIGMA) or Phalloidin-iFluor 647 (ab176759, Abcam), Goat anti-Rabbit Alexa Fluor 488 (A11008, Thermo) and Goat anti-Mouse Cyanine3 (A10521, Thermo). Coverslips were mounted with ProLong™ Gold Antifade Mountant with DAPI (P36940, Thermo).

Peptide Microarrays were screened exactly as described in Li et al (35 (link)); see Supplementary Figure 2 for a flow chart. Mouse primary antibodies were applied at a 1:200 dilution throughout. Their binding was detected using Cy3 labeled Goat anti-mouse IgG (H+L), at 1:300 dilution (A10521, Thermo Fisher (Cramlington, Newcastle, UK). The two quality control antibodies provided by PEPperPRINT, pre-labeled with Cy3 (Polio; KEVPALTAVETGAT) and Cy5 (HA; YPYDVPDYAG), were used at 1:1000 dilution. Two arrays per slide were each treated with 400µl (300µl for PEPperPRINT controls) of the appropriate solutions per well, with slow orbital shaking. Blocking, secondary antibody and control antibody solutions were each incubated at room temperature for 30 min; primary antibody solutions were incubated overnight at 4°C. Arrays were scanned using an Agilent DNA Micro Array scanner with High-Resolution SureScan Technology (Agilent Technologies LDA UK Limited, Stockport, Cheshire ; model G2565CA). The instrument has a dynamic range > four orders of magnitude; by optimizing antibody dilutions the arrays were never saturated while weaker reactivities were still captured. A screengrab of the Agilent image was taken for orientation and editing purposes.