The IHC and ICC

Paraffin-embedded endometrial biopsy sections were subjected to IHC as described previously. Tissue sections were deparaffinized in xylene, rehydrated through a graded series of ethanol, and washed in tap water. For most of the immunostaining, antigen retrieval was performed in a pressure cooker in 10 mM sodium citrate buffer (pH 6.0) for 20 minutes and then the slides were cooled to room temperature. The sections were washed between steps (3 times for 5 minutes each) using 1× phosphate-buffered saline solution containing 0.05% Tween 20 (PBS-T). Nonspecific binding was inhibited by incubating the sections with 10% normal serum for 1 hour at room temperature. After the serum block, sections were incubated overnight at 4°C with the diluted antibody solution in PBS-T containing 1% normal serum.

Labeling was visualized by incubation with a fluorescent-tagged secondary antibody for 1 hour at room temperature. All incubations were done using a humidified chamber protected from light. Slides were mounted using a mounting solution containing DAPI. Pictures were taken using the Olympus BX51 microscope equipped for fluorescent imaging and connected to a Jenoptik ProgRes C14 digital camera with c-mount interface containing a 1.4 Megapixel CCD sensor. Fluorescent images were processed and merged using Adobe Photoshop Extended CS6 (Adobe Systems). HSCOREs were determined as described previously.²³

For ICC analysis of HESC, cells were fixed in 10% (vol/vol) neutral buffered formalin solution for 10 minutes and then washed with PBS. Cells were then permeabilized using PBS containing 0.1% Triton X for 10 minutes at room temperature. Nonspecific binding was inhibited by incubating the sections with 10% normal serum for 1 hour at room temperature. After the serum block, the cells were incubated overnight at 4°C with the diluted antibody solution in PBS containing 1% normal serum. Labeling was visualized by incubation with a fluorescent-tagged secondary antibody for 1 hour at room temperature. One drop of mounting solution containing DAPI was added to each well to stain the nucleus. Pictures were taken using the Olympus Ix70 inverted microscope adapted to a Diagnostic Instrument digital camera containing a 2.0 Megapixel CCD sensor. Fluorescent images were merged and processed using Adobe Photoshop Extended CS6.