Western Blot Analysis of Vaginal IL-33

Vaginal tissues were removed at 0 and 1 dpi, snap frozen in liquid nitrogen, and then homogenized. Homogenized tissue was then lysed in 1mL of cold radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors. Protein concentration was then determined using a Bradford Assay. The immunoblot procedure was then conducted as previously described [72 (link)]. Briefly, 15–20 μg of protein was loaded on to a 10% SDS-Page Gel and run at 95V for 12 min, and 1 hr for 120V. Gels were transferred onto a nitrocellulose membrane for 1 hour at 400 mA. The membranes were then blocked with LI-COR Odyssey blocking buffer (Mandel). Blots were probed for anti-mouse IL-33 (R&D, AF3626) using a 1:1000 dilution and anti-actin (Santa Cruz Biotechnology, sc-1616) at 1:2000 at 4°C overnight. Blots were stripped with LI-COR NewBlot Nitro Stripping Buffer. Primary antibodies were detected using an anti-goat IRDye infrared secondary antibody at a 1:10 000 dilution and then imaged with the LI-COR Odyssey infrared scanner. Band densitometry was assessed using software from Image Studio Lite (LI-COR). IL-33 bands were normalised to actin levels, and then quantified as fold changes to d0 vaginal tissue samples.