Shandon varistain

Uterine and placental samples were fixed in ZSF (for CD4⁺, CD8⁺, and γδ-T cells, NK cells, cytokines IL-10, and tumour necrosis factor-alpha (TNF-α) and C. abortus MOMP antigen) or 4% PFA (for forkhead box P3 (FoxP3)) and were embedded in paraffin wax to create paraffin blocks. The serial sections of each block were cut at 5 μm thickness and mounted onto positively charged adhesive slides (SuperFrost Ultra Plus™, Thermo Fisher Scientific, Braunschweig, Germany). Before starting, the slides were dried overnight at 37 °C. Every sample was identified using permanent markers (CellMark Pen, CellPath Ltd., Newtown, UK). Negative control sections were prepared for each sample substituting the primary monoclonal antibody (mAb) with an IgG isotype control diluted in TBS. Sections of the lymph node tissue were included in every run as positive controls. The slides were warmed up at 60 °C for 30 min and then dewaxed by xylene and rehydrated through graded alcohols (from 100 to 50%) and in distilled water using an automatic processor (Shandon Varistain, Thermo Fisher Scientific, Cheshire, UK).