Immunofluorescence Microscopy of Reproductive Tissues

For immunofluorescence microscopy, tissues were frozen in 2-methylbutane surrounded by dry ice. Frozen blocks were cut into 7 μm sections, fixed in acetone, blocked in a 5% bovine serum albumin PBS solution for 1 h, and stained with DAPI (Invitrogen) and antibodies specific for Thy1.1 (OX-7), CD31 (390) and CD8β (YTS156.7.7) (Biolegend), IFN-γ (XMG12 and CD45.1 (A20) (eBioscience), CXCL9 (goat polyclonal) and CXCL11 (goat polyclonal) (R&D), CX₃CL1 (rabbit polyclonal), ERTR-7, and cytokeratin 8/18 (rabbit polyclonal) (Novus Biologicals), Collagen IV (rabbit polyclonal) (Acris), or CXCL10 (rabbit polyclonal) (Peprotech). IFN-γ-PE staining was amplified using rabbit aντı–PE (Novus Biologicals). Jackson Immunoresearch secondary antibodies conjugated to various fluorochromes were used to stain unconjugated antibodies. Tiled images were acquired with an automated Leica DM5500B microscope and analysis of coronal sections was performed with ImageJ and Adobe Photoshop. Cell isolations and flow cytometry were performed as previously described^{30 (link)}.
Briefly, female reproductive tracts, including the vagina, cervix, uterine horns and ovaries, were excised and chopped into small pieces. Tissue pieces were then digested in 500 mg/L of Collagenase IV (Sigma) while stirring at 300 rpm for 1 h at 37°C with a 1 inch magnetic stir bar. Tissues were then homogenized using a gentleMACS (Miltenyi) and filtered. For chemokine staining by flow cytometry, cells were stained with antibodies specific for CD45.2 (104), CD11c (N418), CD3e (145-2C11) (Ebioscience) and Thy1.1 (HIS51) (BD). Chemokines were stained as previously described^{31 (link)}.