Fix perm solution

For intracellular staining, cells were first stained with ECD-labeled anti-human CD3 (Clone UCHT1, Beckman), FITC-labeled anti-human CD4 (Clone RPA-T4, Biolegend) and then fixed and permeabilized using Perm/Fix solution (Biolegend) at room temperature for 20 minutes. Cells were washed with Perm/Wash buffer (Biolegend) and stained with PE-labeled anti-human IL-17A (Clone BL168, Biolegend). Mouse IgG1 and IgG2 (BD Biosciences) were used as isotype controls. After staining, cells were analyzed with Epics XL (Beckman Coulter) and FlowJo software (Tree Star). Lymphocytes were gated according to forward and side scatter characteristics and CD4⁺T cells were gated based on CD3 and CD4 expression. IL-17 positive lymphocytes, CD3⁺, CD4⁺ T cells were defined by setting regions with the lower limits for cytokine positivity determined from isotype antibody.

A total of 4 mouse uterine tissues (day 11 of pregnancy) were taken from both groups, and mononuclear cells were separated. Mononuclear cells were incubated with anti-mouse CD16/32 (Fc block) for 10mins at room temperature to prevent non-specific binding. Cells were then incubated for 30 mins at room temperature in the following antibody cocktails: CD45-percp-Cy5.5 (Invitrogen, California, USA, 45-0451-82), CD11b-FITC (Invitrogen, California, USA, 11-0112-86) and F4/80-APC (Invitrogen, California, USA, 17-4801-82). The cells were fixed and permeabilized with Perm/Fix solution (BioLegend, USA) and were then stained with LC3B-PE (Cell Signaling Technology, USA, #8899S) after the surface staining. Stained cells were washed twice with PBS and analyzed with a FACS Calibur flow cytometer. We first gated on cells (gate P1) using forward scatter (FSC) and side scatter (SSC), then gated on CD45+ cells (gate P2), then gated on CD11b+F4/80+ cells (gate P3), and analyzed LC3B+ cells in a CD11b+F4/80+ gate. All staining was performed in accordance with manufacturer’s protocols. Isotype controls were used to confirm antibody specificity. Single colour stain controls were used to enable correct compensation.