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Midguts and washed salivary glands were placed in separate drops of PBS on silane-treated glass slides (BioWorld, Dublin, OH), dried, and fixed with acetone for 10 min. To block nonspecific binding the slides were incubated with 10% bovine serum albumin (BSA) in PBS at room temperature for 30 min, and then stained with fluorescein isothiocyanate (FITC -conjugated rabbit anti-B.

To block non-specific antibody binding, the slides were incubated with 10% goat serum in PBS for 30 minutes.

To block aspecific binding the slides were incubated with Tris-buffered saline (TBS) supplemented with 5% goat serum.

After antibody binding, the slides were processed for colour development using the avidin biotinylated peroxidase method, and were counterstained with Mayer's haemotoxylin.

Endogenous peroxidase activity was blocked with 3%H2O22 in methanol for 10 min. After pretreatment with Tris-EDTA buffer, in order to block non-specific antibody binding, the slides were incubated with 10% goat serum in PBS for 30 min. Primary monoclonal ERα antibody (ER-6F11, Novocastra, Newcastle, UK) was used at 1 50 dilution and detected following standard immunohistochemical techniques.

After blocking non-specific binding sites, the slides were reacted with the appropriate dilution of hypoxyprobe™-1 Mand (1 : 500) and rabbit anticleaved caspase-3 (Asp175; Cell Signaling Technology; 1 : 1000) overnight at 4°C.

To saturate unspecific binding sites, the slides were incubated with washing buffer containing 10% donkey serum (blocking buffer) or 0.5% BSA for 30 min at room temperature.

To inhibit nonspecific binding of antibodies, the slides were treated with 3% normal goat serum before the overnight incubation of the slides with anti-CARM1 antibodies.

After nonspecific binding was blocked, the slides were incubated with NDRG2 antibody (1 200; Abnova, Taipei, Taiwan) or GLUT1 antibody (1 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in phosphate-buffered saline (PBS) at 4°C overnight in a humidified container.

In order to block the nonspecific binding of the antibody, the slides were incubated for 30 min with 5% bovine seroalbumin (BSA) in PBS at room temperature.

To block nonspecific binding of the secondary antibodies, the slides were incubated with 4% donkey serum diluted in blocking buffer for 30 min.