Anti involucrin antibody

SCC13 cells (1 × 10⁶ cells) were admixed with HDFs (5 × 10⁵ cells) with shRNA-mediated silencing of PDCD4 or control in 150 μl of growth factor-reduced matrigel (BD Bioscience) and intra-dermally injected into the back skin of 6-week-old NOD/SCID mice (Taconic Farms Inc.) as previously described [30 (link)]. For the ear injection, SCC13 cells (1 × 10⁵ cells) were admixed with equal numbers of HDFs with shRNA-mediated silencing of PDCD4 or control. Cells were resuspended in 3 μl of Hanks’ balanced salt solution and then injected intradermally into the left and right tip of the ear dermis of 10-week-old NOD/SCID mice using a Hamilton microsyringe fitted with 33 gauge needle as performed in [3 (link)]. Mice were sacrificed 3 weeks after injection and tumors were removed for analysis.
Tissue immunofluorescence was performed as before [2 (link), 3 (link)]. Anti- ki67 antibody, anti-Vimentin antibody (Abcam), anti-p63 antibody (Santa Cruz Biotechnology), anti-Involucrin antibody (Sigma), anti-Periostin antibody (Abcam), anti-Tenascin C antibody (Santa Cruz Biotechnology), and anti-αSMA antibody (Santa Cruz Biotechnology) were used. Quantification of all images of tissue immunofluorescence staining was performed using ImageJ and Adobe Photoshop software.
All animal studies were performed following the approved Institutional animal protocol procedure (IACUC-MGH).

HPV11.HaCaT cells were cultured overnight on glass slides, which were in 3 cm petri dishes. The cultures were rinsed three times with PBS and fixed in 4% paraformaldehyde solution. 1ml 30% triton-X-100 was added in 299ml TBS to compound scrubbing solution. Subsequently, they were washed and then blocked by goat serum for 1h at room temperature. Then incubated overnight at 4°C in anti-HPV11 E7 antibody (1:250 dilution in blocking buffer; Abcam, USA) or anti-involucrin antibody (1:200 dilution in blocking buffer; Sigma-Aldrich, USA), washed three times for 5 min each time, followed by incubating in goat anti-mouse IgG-conjugated with Alex Fluor 488 (1:400 dilution in PBS; Beyotime, China) for 1 h at 37°C in dark. DAPI solution (3 μg/mL in PBS; Beyotime, China) was used for nuclear staining. Samples was observed under a laser scanning confocal microscope (Olympus, Japan). In the fluorescent images, cytoplasm displayed as green fluorescence and the nucleus displayed as blue.