Cd163

Data collection and analysis were performed blind to the conditions of the experiments. FFPE sections were blocked and permeabilized with PBS containing 5% normal donkey serum and 0.5% Triton-X 100 for 1 h at room temperature (RT). Primary antibodies were incubated overnight with combinations of Iba1 (WACO), Iba1 (Synaptic Systems), MRC1 (also known as CD206) (Abnova), CD1C (Abcam), CD163 (Sigma-Aldrich), S100A6 (Sigma-Aldrich), SIGLEC1 (also known as CD169) (Sigma-Aldrich) and collagen IV (Sigma-Aldrich,). Secondary antibodies were added as follows: Alexa Fluor 488 1:500 dilution (Thermo Fisher Scientific), Alexa Fluor 568 1:500 dilution (Thermo Fisher Scientific), Alexa Fluor 647 1:500 dilution (Thermo Fisher Scientific) and Alexa Fluor 647 (Jackson ImmunoResearch Laboratories) 1:500 dilution for 2 h at RT. Nuclei were counterstained with DAPI (Carl Roth) when necessary. Images were taken using conventional fluorescence microscopes (Olympus BX-61 and Keyence BZ-9000) and confocal pictures were taken with a Leica TCS SP8 (Leica). Image quantification was conducted in Adobe Photoshop. Outlier values were identified using Grubbs’s test in R and removed.

The immunofluorescence staining was carried out on paraffin placenta sections, which were dewaxed for 20 min in Roticlear (Roth, Karlsruhe, Germany). They were then dehydrated in 100%, 70%, and 50% ethanol (CLN GmbH, Freising, Germany) to distilled water. To unmask the proteins, the slides were treated with sodium-citrate-buffer in a pressure cooker. The blocking was carried out for 15 min with the help of UltraVision-Protein-Block (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies CD68 (Sigma Aldrich, Saint Louis, MO, USA), CD80 (Sigma Aldrich, Saint Louis, MO, USA), and CD163 (Sigma Aldrich, Saint Louis, MO, USA) were diluted in dilution medium (DAKO, Aligent Technologies, Santa Clara, CA, USA), applied to the sections and incubated in the fridge at 4 °C for 16 h. Then the two secondary antibodies Goat-Anti-Rabbit Cy3 (Dianova, Hamburg, Germany) and Goat-Anti-Rabbit Alexa Fluor 488 (Dianova, Hamburg, Germany) were also mixed together in dilution medium and incubated for 30 min at RT in the dark. Finally, the slides were dried and covered with mounting medium for fluorescence with DAPI (Vector Lab, Burlingame, CA, USA). To ensure that the staining did not stain unspecifically, a negative control with an IgG antibody was also carried out.