**2.5.4 Immunohistochemistry**

3-5 µm thick sections obtained from formaldehyde fixed, paraffin-embedded tissue were dehydrated in graded ethanol. After antigen retrieval, slides were blocked using 3% BSA in PBS and incubated with mouse anti-human αvβ3 integrin (R&D Systems, Minneapolis, MN, USA), Muc-1 (Abcam, Cambridge, UK), LIF and rat anti-human MECA-79 monoclonal antibody (Santa Cruz biotechnology, INC., Santa Cruz, California, USA). Excess primary antibody was washed with PBS and the sections were again incubated with anti-mouse and anti-rat biotinylated secondary antibody (Santa Cruz biotechnology, INC., Santa Cruz, California, USA) according to the manufacturer's protocol, before incubation with avidin biotinylated horseradish peroxidase (Santa Cruz biotechnology, INC., Santa Cruz, California, USA). Labeled cells were visualized with Diaminobenzidine (DAB) and sections counterstained with hematoxylin. Next, the slides were dehydrated using series of alcohol gradient and mounted using distrene, tricresyl phosphate (DPX) and xylene. The slides were then examined under bright field microscope (Carl Zeiss, Jena, Germany).

#### **2.5.5 Scanning electron microscopy**

Formaldehyde-fixed tissues were washed in PBS and dehydrated in a series of alcohol gradient (50%, 70%, 90%, 95%, 100%), each for 10 mins, dipped in HMDS (1,1,1,3,3,3 hexamethyl disilazane; SRL, Bombay, India) and air dried. Dried tissues were then mounted and coated with gold and the luminal endometrial surface thoroughly examined under SEM (Jeol JSM-5800 Scanning Microscope, Tokyo, Japan). Pinopode formation was also evaluated semi-quantitatively depending on their stage of development on the surface of the endometrium, and scored as (i) well-developed (ii) poorly developed and (iii) absent and on their abundance and scored as (i) abundant (ii) moderate (iii) few.

#### **2.5.6 Real-time PCR**

Levels of COX-2, MMP-2, -9, TIMP-1 and -2 gene expression were analyzed by real time PCR (RT-PCR), which was performed with ABI Prism 7000 Sequence Detection System (Applied Biosystems Inc., Carlsbad, California, USA) using syber green master mix (Applied Biosystems Inc., Carlsbad, California, USA). RT-PCR primers were designed using sequence data. Total RNA was isolated from tissue by RNA isolation kit (Trizol Reagent, Invitrogen, Carlsbad, California, USA) and 10 μl of total RNA isolated was subjected to reverse transcription for cDNA synthesis with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Carlsbad, California, USA), according to the manufacturer's instructions. After synthesis, 5 μl of cDNA was used for the RT-PCR mixed with syber green. At the end of each reaction, Cycle threshold (Ct) was manually set at the level that reflected the best kinetic PCR parameters, and melting curves were acquired and analyzed. Relative quantification was used to measure gene expression by relating the PCR signal.
