Hoechst 33342 nuclear

Cultured cells were fixed with methanol for 30 min, and immunostained with acetyl-p53 (1:500, Cell Signaling, #2570), p-p53 (1:500, Cell Signaling, #9286), or NF-κB p65 (1:1000, Cell Signaling, #8242) for 4 hours at 4 °C, followed by incubation with Alexa Fluor-488 or −594 secondary antibodies (1:1000, Molecular Probes, Life Technologies, A11034 and R37121) for 2 h at 25 °C, and by Hoechst 33342 nuclear staining (1:2000, Sigma, #23491-52-3) for 15 min. Imaging of 96-well plate was performed using a high-content imaging system (MetaXpress Ultra, Molecular Devices) with ×40 objective. Airyscan super-resolution confocal images were acquired with LSM800 (Zeiss Germany) with ×63 objective. Image analyses were conducted using the Color Deconvolution Plugin in ImageJ software (NIH; 1.51n)^{69 (link)}.

Following antigen retrieval with citrate buffer (0.01 M, pH 6), 5 μm cryosections of primary tumours or lymph nodes were fixed with 4% paraformaldehyde (Sigma) and then incubated with one or more primary antibodies for 16 h at 4 °C. Anti-LYVE-1 (Fitzgerald, catalogue #: 70R-LR003, 1:500) antibody was used to localize lymphatic vasculature followed by incubation with corresponding Alexa-Fluor-conjugated secondary antibody (Invitrogen, catalogue #: A31573, 1:500) and Hoechst 33342 nuclear staining (Sigma, catalogue #: B2261, 1:1000). Immunostaining was visualized at × 40 magnification using an Eclipse 90i microscope (Nikon). LYVE-1⁺ LVD was evaluated in ⩾5 digital images per sample captured at random intervals around primary tumour margins32 (link) by an investigator blinded to treatment conditions. Area of vessel density (percentage of total tissue area) was quantified using ImageJ software.