Ab211926

Sections with a thickness of 4 μm were cut, and slides were deparaffinized and rehydrated. The sections were pretreated and stained using the Omnis automated slide-processing system from Agilent (Glostrup, Denmark). The tissue sections were subjected to heat-induced epitope retrieval pretreatment using EnVision™ FLEX Target Retrieval Solution High pH (GV804, Dako Omnis, Agilent) for TIMP-1 or EnVision™ FLEX Target Retrieval Solution Low pH (GV805, Dako Omnis, Agilent) for collagen I for 30 min, followed by incubation with rabbit monoclonal antibodies against TIMP-1 (clone EPR18352, 1:500, ab211926, Abcam, Cambridge, UK) or collagen α1 (I) (E8F4L, 1:100, #72026, Cell Signaling, Danvers, MA, USA) for 30 min at 32 °C. The reactions were detected using the standard polymer technique EnVision™ FLEX/HRP Detection Reagent (GV800, Dako Omnis, Agilent), and signal intensity was enhanced using the EnVision™ FLEX+ Rabbit LINKER (GV809, Dako Omnis, Agilent) and visualized using EnVision™ Flex DAB+ Chromogen system (GV825, Dako Omnis, Agilent). Finally, the sections were counterstained with hematoxylin and mounted with Pertex. The immunostained sections were evaluated by a senior consultant pathologist (L.M.R.G.).

The tissue sections were incubated with anti-CHI3L1 (abcam, ab255297, 1:250), anti-MSN (abcam, ab151542, 1:250) or anti-TIMP1 (abcam, ab211926, 1:250) primary antibody overnight at 4° C. After washing three times, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 20 minutes, followed by staining with diaminobenzidine. Finally, the sections were counterstained with hematoxylin.

MMP-9 and TIMP-1 were assessed from menstrual blood through immunocytochemistry. To assess MMP-9 and TIMP-1 in menstrual blood, 1 mL (20 drops) of menstrual blood was collected during the 2nd or 3rd day of the menstrual cycle and diluted into 20 ml of preservative. Samples were incubated at room temperature for 30 min and centrifuged at 744× g for 15 min. The precipitate was diluted and transferred to a glass slide. Following antigen retrieval, sections were incubated in 0.3% H₂O₂ (30% stock H₂O₂ diluted in methanol) and washed in PBS for 2 × 5 s. Sections were incubated in blocking reagent for 2 × 5 minutes, after which the blocking reagent was tipped off the slide, and a primary antibody (anti-MMP9 antibody, 1:1000, ab38898, Abcam; recombinant anti-TIMP-1 antibody, 1:1000, ab211926, Abcam) was applied. Sections were incubated in primary antibody for 1 hour, washed in PBS for 3 times, 5 s each time, and incubated in secondary antibody (R.T.U Biotinylated Universal Secondary Antibody BP-1400, Vector), for 2 times, 5 s each time. Color was developed using chromogen 3,3′-diaminobenzidine (DAB), and the slides were counterstained using Mayer’s hematoxylin (Sigma). Sections were dehydrated in 70%, 80%, and 90% industrial methylated spirit (IMS) for 3 min each, and mounted in xylol for 3 min.

The method was described in our previous publications 23 (link). The primary antibodies used were as follows: ESM1 (Abcam, ab103590 at 1/800 dilution), Akt (Abcam, ab8805 at 1/500 dilution), p-AKT (Abcam, ab38449 at 1/1000 dilution), mTOR (Abcam, ab245370 at 1/2000 dilution), p-mTOR (Abcam, ab109268 at 1/2000 dilution), MMP9 (Abcam, ab76003 at 1/2000 dilution), TIMP (Abcam, ab211926 at 1/1000 dilution), eNOS (Abcam, ab252439 at 1/1000 dilution), HIF-1α (Abcam, ab51608 at 1/500 dilution) and GAPDH (Abcam, ab181602 at 1/10000 dilution).

Transfected cells were lysed with RIPA lysis buffer (Thermo, U.S.A.) containing protease inhibitors (Thermo, U.S.A.). The concentration of extracted protein was measured by using a BCA protein assay kit (Thermo, U.S.A.). Equal amounts of protein were separated with 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, U.S.A.). The membranes were then blocked with 5% non-fat milk in TBST for 1 h at room temperature, followed by incubation with primary antibodies of PIK3R3 (ab238509), cyclin D1 (ab16663), cyclin E1 (ab33911), CDK2 (ab32147), CDK4 (), p21 (ab109520), p27 (ab32034), MMP-2 (ab92536), MMP-9 (ab76003) and TIMP-1 (ab211926) (Abcam, U.S.A.) overnight at 4°C. Subsequently, the membranes were washed with TBST three times and probed with the corresponding horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology Inc., U.S.A.) for 2 h at room temperature. ECL reagent (Pierce) was used to detect the signals on the membranes.