Mouse anti pan cytokeratin

Heat-fixed paraffin-embedded sections were processed for immunofluorescence analysis as previously described [46 (link),47 (link)]. Slides were incubated overnight at 4°C with one of the following antibody combinations diluted in blocking buffer (10% normal donkey serum, 1% bovine serum albumin in PBS-T): mouse anti-pan-cytokeratin at 1:150 (Cell Signaling 4545) and rabbit anti-vimentin at 1:100 (Cell Signaling 5741); rabbit anti-Keratin 5 at 1:1000 (CAT#: PRB-160P, Covance), or mouse anti-cytokeratin 8 (CAT#: NB120-9287, Novus). After sections were washed with PBS-T, Invitrogen Alexa Fluor 488 or 594 anti-mouse or anti-rabbit IgG secondary antibodies at 1:150 were applied for 1 hour at room temperature in the dark. After serial washes with PBS-T, sections were incubated with 20 μg/mL Hoechst 33342 in PBS for 10 min to visualize nuclei. Coverslips were mounted using PermaFluor Mountant media (CAT#: TA-030-FM, Thermo). Slides were viewed using a Leica 6000 epifluorescent microscope and digital images were captured using Leica LAS software.

Frozen endometriotic lesions were embedded in optimal cutting temperature compound and sectioned at a 5 μm thickness. Then, the sections were washed with PBS, permeabilized with 0.2% Triton X-100, washed with PBS for 10 minutes, and blocked with 10% FBS for 1 hour. The sections were then incubated with primary antibodies overnight at 4°C, washed with PBST, and then, where required, stained with Alexa Fluor–conjugated secondary antibodies for 1 hour at room temperature. The following primary antibodies were used: Goat Anti-human IL-33 (1:40, R&D Systems, AF3625), Mouse Anti-human Vimentin (1:400, Cell Signaling Technology, clone D21H3), Mouse Anti-Pan cytokeratin (1:40, Cell Signaling Technology, clone AE1/AE3), Rabbit Anti-goat Alexa Fluor 647 (1:200; Invitrogen, A27018). Immunostained tissue sections were imaged using the Leica TCS SP8 confocal microscope. Images were taken in a Z-stack (1.20 μm; Z dimension) and tile scan (12 tiles, 580 μm × 435 μm each tile; x and y dimensions) acquisition mode. Two to 3 fields per preparation were imaged using HC PL APO CS2 ×63/1.40 oil objective and LAS-X Software (Leica Microsystems).