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).
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).
