Endogenous avidin biotin blocking kit

E12.5, E13.5 and E14.5 mouse embryos were proceeded for paraffin-embedded classical histology and were sectioned into 5-µm thickness. The sections were mounted on positively charged slides. The slides were deparaffinized using Histolemon and re-hydrated. Epitopes retrieval was performed by incubation in 0.05% citraconic anhydride buffer (pH 7.4) at 100  °C for 15 min. Endogenous peroxidase was inhibited by incubation in 30% H₂O₂ for 20 min. Endogenous biotin molecules were blocked with endogenous avidin/biotin blocking kit (ab64212), and non-specific binding was blocked by incubation with 10% goat serum diluted in PBS for 1 h. Subsequently, the sections were incubated overnight in a humid chamber at RT 1 h with ERα antibody (1/200; ab32063 abcam). Sections were extensively washed and incubated for 1 h with the biotinylated anti-rabbit IgG secondary antibody (1/500; ab97049 abcam). Sections were extensively washed and incubated 1 h with HRP-conjugated streptavidin (1/1000; ab7403 abcam). The sections were finally treated with diaminobenzidine in the dark, washed, then rapidly counterstained with Mayer’s Hemalun and mounted in Eukitt medium.

Sections were deparafinized in Xylene and Ethanol and rinsed in water. Heat‐induced epitope retrieval with Tris‐EDTA, pH = 9 was performed for 2.5 h in a steamer. Sections were washed in TBS, permeabilized with 0.2% Tween, washed, and blocked for 1 h in 5% BSA (Jackson Immunoresearch, Hamburg, Germany) in TBS. An endogenous avidin/biotin blocking kit (Abcam) was used if biotin‐streptavidin amplification was employed. The same primary antibodies were used for FFPE as for frozen sections, aside from the mouse anti‐perforin antibody (5B10, dilution, 1:30; Dianova, Hamburg, Germany) and the rabbit anti‐CD163 antibody (EPR19518, dilution 1:200; Abcam). Isotype control stainings were performed to ensure specificities.