Protein block serum

Paraffin-embedded tissues were sectioned to 5 µm thickness using a microtome. Uterine sections were deparaffinized and rehydrated. Endogenous peroxidase was inactivated with 3% H₂O₂. Sections were subjected to antigen retrieval in 0.01 M sodium citrate buffer (pH 6.0). Nonspecific staining was blocked using protein block serum (Dako, Carpinteria, CA, USA) for 1 h. Sections were incubated with primary antibodies at 4 °C overnight. On the following day, sections were incubated with appropriate secondary antibodies for 1 h at room temperature. Sections were counterstained using Topro-3-iodide (TOPRO; Life Technologies, Carlsbad, CA, USA) and mounted. For immunohistochemistry, DAB reagent (Vector Laboratories, Inc., Burlingame, CA, USA) was applied to visualize signals. Images were observed under a microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using ZEN software (Carl Zeiss).

Female reproductive organs were dissected, and then fixed in 4% paraformaldehyde for histology or snap frozen for RNA and/or protein preparation. Fixed tissues were washed, dehydrated, and embedded in Paraplast (Merck KGaA, Darmstadt, Germany). Paraffin-embedded tissues were sectioned using a microtome, stained with hematoxylin and eosin (H&E) (Sigma-Aldrich), and observed by a light microscopy.
For immunostaining analyses, antibodies specific to PR (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA, 1:100), α-SMA (Abcam, Cambridge, UK, 1:100), and Ki-67 (Abcam, 1:100) were used in 5 μm thick paraffin-embedded sections. Blocking was carried out using protein block serum (Dako, Carpinteria, CA, USA) for 1 h, and then samples were incubated with an appropriate primary antibody at 4 °C overnight. On the following day, sections were washed in PBS and incubated with HRP secondary antibody or FITC conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. Nuclear staining was performed using TO-PRO-3-iodide (Life Technologies, Carlsbad, CA, USA). For Immunohistochemistry, DAB reagent (Vector Laboratories, Inc., Burlingame, CA, USA) was applied to visualize signals. Slides were counterstained with hematoxylin, mounted with mounting solution and cover-slipped.

To determine the expression of COL1A1 (1:200; NB600-408; Novus Biologicals) (Jun et al., 2019 (link)) after uterine damage, frozen uterine sections (12 μm) were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature, washed in PBS, and treated with 0.1% Triton X-100 for 10 min at room temperature and washed in PBS. Non-specific staining was blocked using protein block serum (Dako, Carpinteria, CA, United States). The sections were then incubated overnight with primary antibody at 4°C, washed in PBS, and incubated with secondary antibody (A-11001, 1:1000; Invitrogen) for 60 min at room temperature. After three washes in PBS, the sections were counterstained and mounted. Images were obtained using a suitable microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using ZEN software (Carl Zeiss).