Sc 810

After dewaxing, tissue sections were soaked in sodium citrate buffer, placed in an autoclave for antigenic thermal repair and pretreated with 0.3% H₂O₂ in methanol to quench the endogenous peroxidase activity. Subsequently, the sample was incubated with 5% bovine serum albumin (BSA) at 37°C 30 min for blocking. Primary antibodies, including anti-α-smooth muscle actin (α-SMA; 1:1,000; 80008-1-RR; Proteintech Group, Inc.), anti-mucin 1 (MUC-1; 1:100; MA5-35250; Thermo Fisher Scientific, Inc.), anti-estrogen receptor (ER; 1:100; 21244-1-AP; Proteintech Group, Inc.), anti-progesterone receptor (PR; 1:100; sc-810; Santa Cruz Biotechnology, Inc.) and anti-cytokeratin 19 (CK19; 1:200; 10712-1-AP; Proteintech Group, Inc.), were used to incubate the tissue overnight at 4°C. After the primary antibody was fully reacted, the secondary antibody was applied and the subsequent steps were carried out according to the instructions of the immunohistochemistry kit (KIT-9710; Fuzhou Maixin Biotech Co. Ltd.), followed by 3,3′-diaminobenzidine development and hematoxylin staining for differentiation, and microscopic observation of the blocked slices.

A portion of each endometrial sample was fixed in paraformaldehyde, included in paraffin blocks and 5 μm sections were prepared. PR-A/B, PR-B, glycodelin and Specificity protein 1 (Sp1) were evaluated by IHC in the endometrial samples using the antibodies and dilutions shown in Table 
2 and the broad spectrum Histostain-SP kit (Life Technologies, Carlsbad, CA, USA) as described previously
[21 (link)]. Immunoreactive PRA/B, PRB, Sp1 and glycodelin in endometrial sections was semi-quantified using the expression level score (ELS), calculated by means of Image Pro Plus software (Media Cybernetics Rockville, MD, USA) as described previously
[21 (link)]. Briefly, ELS = Mean Optical Density of immunostaining x Percent Area Positively Stained x 100.Table 2

Antibodies and dilutions used for immunohistochemistry

Antibody	Source	Dilution
Progesterone receptor (PR)-A/B	Santa Cruz Biotech. (sc-810)	1:50
PR-B	Novocastra (NCL-PGR-B)	1:100
Glycodelin	H. Koistinen [22 (link)]	1:1000
Specificity protein-1 (Sp1)	Santa Cruz Biotech. (sc-14027)	1:100

Primary anti-VEGFA (ab52917, Abcam, USA), anti-vWF (PB0273, Boster, Wuhan, China), anti-PCNA (BM0104, Boster, Wuhan, China), anti-LC3 (WL01506, Wanlei, Shenyang, China), anti-p62 (WL02385, Wanlei, Shenyang, China), anti-ERα (ab32063, Abcam, USA), and anti-PR (sc-810, Santa Cruz, TX, USA) antibodies were diluted in 0.5% goat serum in PBS. The sections were heated in a microwave in sodium citrate solution for antigen recovery and pretreated with 0.3% H₂O₂ in methanol to quench endogenous peroxidase activity. Then, the samples were incubated with 3% goat serum to block nonspecific antibody binding sites. Subsequently, the samples were incubated with primary antibodies at 4 °C overnight. Immunoreactivity was visualized using a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab64264, Abcam) according to the manufacturer’s instructions.