**2.5.7 Western blotting**

330 Endometriosis - Basic Concepts and Current Research Trends

Endometrial tissue was first digested in 2% collagenase-1 (Invitrogen, Grand Island, NY, USA) in DMEM (Himedia, Mumbai, India) for 1.5 to 2 hrs at 37°C and then centrifuged to isolate the stromal cells. Undigested glands were then treated with 0.25% trypsin-0.02% EDTA (Himedia, Mumbai, India) for 4–8min, washed with 10% FBS-DMEM. Single epithelial cells were isolated by centrifugation, as described previously. Isolated cells were washed, RBC lysed using RBC lysis solution and fixed in 2% paraformaldehyde (20 min at RT). Single cell suspension thus obtained was divided into five parts; four parts were stained with mouse anti-human αvβ<sup>3</sup> integrin, LIF (R&D Systems, Minneapolis, MN, USA), Muc-1 (Abcam, Cambridge, UK) and rat anti-human MECA-79 monoclonal antibody (Santa Cruz 1 Biotechnology, Inc., Santa Cruz, California, USA) according to instructions provided by the manufacturer in the manual. The fifth part remained unstained. Excess antibodies were washed out and the cells again incubated with fluorescein conjugated secondary goat anti-mouse and anti-rat IgG (R&D Systems, Minneapolis, MN, USA). After washing excess antibodies, the stained cells were analyzed using flow cytometer (BD FACS Calibur™, BD Biosciences, San Jose, CA, USA).

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

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

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

then examined under bright field microscope (Carl Zeiss, Jena, Germany).

their abundance and scored as (i) abundant (ii) moderate (iii) few.

**2.5.3 Isolation of cells and flow cytometric analysis** 

**2.5.4 Immunohistochemistry** 

**2.5.5 Scanning electron microscopy** 

**2.5.6 Real-time PCR** 

The endometrial tissue was homogenized in tissue lysis buffer. The tissue lysate was then centrifuged at 15,000 g for 15 min and the protein concentration of the homogenates was determined by the GeNei™ Protein Estimation Kits (Bangalore Genei, India). 30 µg of homogenate protein were separated by SDS-polyacrylamide gel electrophoresis (SDS- PAGE). The separated proteins were electroblotted onto a Hybond PVDF membrane (GE Healthcare) at 30 volt for 13 hrs. After blocking the non-specific binding sites with non-fat dry milk in TBST buffer for 1 hr at room temperature, the blots were incubated overnight at 4°C with rabbit polyclonal antibody against COX-2, mouse monoclonal antibody against MMP-2,-9, TIMP-1 and -2 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), rabbit polyclonal antibody against VEGF, VEGFR1+VEGFR2 (Abcam, Cambridge, UK). The blots were then washed three times with TBST buffer, incubated for 1 hr at room temperature with horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). After further washing, the immunoreactive proteins were revealed using the DAB as substrate.

### **2.5.8 Statistical analysis**

Data were compared using independent two sample't' test and chi-square test, as applicable. Ky Plot version 2.0 beta 13 software and Graphpad Prism Software were used for this purpose. Statistical significance was defined as p≤0.05.
