Vectorshield hard set mounting medium

Cells grown on glass coverslips were fixed with 4% formaldehyde for 15 min at room temperature, rinsed three times in PBS plus 0.3% Triton for 5 min and blocked in 5% BSA for 30 min at room temperature. Cells were then incubated with anti-FoxK1 antibody (Abcam ab18196) and secondary antibody coupled to AlexaFluor 488 (Life Technologies). Cells were mounted on glass slides using Vectorshield hard set mounting medium (Vector Laboratories). Images were acquired with a two-photon confocal microscope (Zeiss 710). For EdU incorporation assay, cells were treated with 10 μM EdU (8 h) and stained with Click-iT Plus EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher).

For IHC, the tissues were fixed in neutral buffer with 10% formalin and then embedded on slides. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. The samples were then pretreated with Borg Decloaker, and blocked in background Sniper solution. After washing, the samples were incubated with specific primary antibodies for CMAH (Santa Cruz; Texas, USA, 1∶100) and Neu5Gc (Sialix; San Diego, CA, USA; 1∶200) at 4°C overnight. After the incubation, the samples were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Samples were then stained with ImmPACT™ DAB peroxidase substrate (Vector Laboratories; CA, USA) to visualize the signal. Samples were also stained with Hematoxylin QS to provide background information for reference. The samples were mounted using VECTORSHIELD HardSet mounting medium (Vector Laboratories; CA, USA) and observed using fluorescence microscopy (Olympus; Japan).