Nbp2 24891

Immunohistochemistry for PPARD, CYP21A2, IFNαR1, and IFNαR2 in the bovine CLs on day 18 of pregnancy and on day 15 of non-pregnancy was performed using the automated Ventana HX System
Discovery with a DabMapKit (Roche Diagnostics) as per a previously described method [15 (link)]. The 5 µm-thick sections from the paraffin-embedded luteal
tissue were incubated at room temperature (20˚C) with rabbit polyclonal anti-human PPARD antibody (#ARP38765_T100, Aviva Systems Biology, San Diego, CA, USA; 1:50), rabbit polyclonal
anti-human CYP21A2 antibody (#ARP60144_P050, Aviva Systems Biology; 1:25), rabbit polyclonal anti-human IFNαR1 antibody (#bs-4116R, Bioss, Woburn, MA, USA; 1:50) or rabbit polyclonal
anti-human IFNαR2 antibody (#bs-7022R, Bioss; 1:50) for 12 h. The signals were detected using anti-rabbit IgG-Biotin conjugate (Sigma-Aldrich, LLC, St Louis, MO, USA; 1:100) for 1 h, and
then counterstained with hematoxylin. Negative controls were assessed using normal rabbit IgG (#NBP2-24891, Novus Biologicals LLC, Littleton, CO, USA) diluted at concentrations equivalent to
the primary antibodies. The sections were observed with a Leica DMRE HC microscope (Leica Microsystems, Tokyo, Japan) and photographed with a Nikon Digital Sight DS-Fi1-L2 (Nikon
Instruments, Tokyo, Japan).

Immunohistochemistry for CCL8, CCR1, CCR2, and CCR5 in the bovine placentome at parturition was performed using the automated Ventana HX System Discovery with a DabMapKit (Roche
Diagnostics, Basel, Switzerland), as described previously [16 (link)]. The 5 µm-thick sections from formalin-fixed and paraffin-embedded tissue were incubated
at room temperature with rabbit polyclonal anti-bovine CCL8 antibody (MBS2026335; 1:400; MyBioSource, San Diego, CA), rabbit polyclonal anti-human CCR1 antibody (ab140756; 1:200; Abcam PLC,
Cambridge, UK), rabbit polyclonal anti-human CCR2 antibody (NBP1-48337; 1:100; Novus Biologicals LLC, Littleton, CO), or rabbit polyclonal anti-human CCR5 antibody (NBP2-31374SS; 1:100;
Novus Biologicals LLC) for 12 h. The signals were detected using anti-rabbit IgG-biotin conjugate (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:100 for 1 h and then counterstained with
hematoxylin. Negative controls were performed using normal rabbit IgG (NBP2-24891; Novus Biologicals LLC) diluted at concentrations equivalent to the primary antibodies.

Immunohistochemistry for CCR3 and XCR1 in the bovine CL on days 150–160 of pregnancy was performed using the automated Ventana HX System Discovery with a DabMapKit (Roche Diagnostics GmbH, Mannheim, Germany) as described previously in detail by our laboratory [21 (link)]. The CL samples for immunohistochemistry were fixed with 10% neutral formalin for 24 h. The 5 µm-thick sections from paraffin embedded luteal tissue were incubated at room temperature with a rabbit polyclonal anti-human CCR3 antibody (251536, Abbiotec LLC, San Diego, CA, USA) or rabbit polyclonal anti-human XCR1 antibody (LS-A159, LifeSpan BioSciences, Seattle, WA, USA) diluted 1:100 each in Discovery Ab diluents (Roche) for 2 h. Nucleotide and amino acid sequence homologies of human CCR3 with bovine CCR3 are 80% and 74%, and those of human XCR1 with bovine XCR1 are 85 and 78%, respectively. The signals were detected using anti-rabbit IgG-Biotin conjugate (Sigma-Aldrich,
St Louis, MO, USA) diluted 1:100 for 1 h and then counterstained with hematoxylin. Negative controls were performed using normal rabbit IgG (NBP2-24891, Novus Biologicals, LLC, Littleton, CO, USA) diluted at concentrations equivalent to the primary antibodies. The sections were observed with a Leica DMRE HC microscope (Leica Microsystems, Tokyo, Japan) and a Nikon Digital Sight DS-Fi1-L2 (Nikon Instech, Tokyo, Japan).