X81 fluorescence microscope

At designated time point, mice were perfused with 4% polyformaldehyde (PFA) (20–30 mL) before dissection of brain tissue. The brain tissue was dehydrated with 30% sucrose 24 h before being embedded in Tissue Tek OCT (Sakura Finetek), followed by snap freezing in liquid nitrogen. The OCT embedded tissue blocks were kept at −80 °C for cryosection. The obtained brain slices (15 µm-thick) were fixed in Z-fix solution (Anatech) for 10 min, and permeabilized with 0.1% Triton X-100 for 5 min. To stain the mature neuron cells at the periphery of TBI site, a mature neuron marker, NeuN, was chosen. Anti-NeuN primary antibody was obtained from Cell Signaling Technology (1:150, Cat# 12943). The Cy3-conjugated donkey anti-rabbit secondary antibody (1:200, Cat# 711165-152, Jackson ImmunoResearch) was used. The nuclei were counterstained with DAPI using Vectashield mounting medium (Vector Lab). The images were taken from Olympus X81 fluorescence microscope.

HKc/HPV16-Ctrl and HKc/HPV16-Six1 were plated in Lab-Tek II chambers (Nalge Nunc International, Rochester, NY) for 24 h. Following treatment, fixation (with 2% paraformaldehyde in PBS, pH 7.2) and permeabilization (with 0.1% Triton X-100 in PBS), samples were incubated with antibodies against E-cadherin (at 1:200 dilution), fibronectin (at 1:200 dilution) and keratin 10 (at 1:100 dilution) overnight at 4°C. Samples were then washed three times with PBST, followed by incubation with FITC- and Alexa 568- conjugated secondary antibodies (at 1:1000 dilution, Invitrogen). Nuclei were stained with a 1:20,000 dilution of 4’, 6-diamidino-2-phenylindole (DAPI) (Invitrogen) before cells were mounted. Samples were observed using an Olympus X81 fluorescence microscope.