Avidin biotin peroxidase

Human tissue sections were deparaffinized and hydrated by a series of xylene and alcohol treatment. The sections were incubated with rabbit polyclonal anti-MPC1 (Abcam, Cambridge, UK), and mouse polyclonal anti-HIF1α (Abcam) antibodies at 4 °C overnight, followed by incubation with avidin-biotin-peroxidase (DAKO). MPC1 and HIF1α were considered positive by cytoplasmic staining and the expression levels were semi-quantitatively analyzed using a composite score system by assessing both the percentage and intensity of stained tumor cells. The percentage of positive cells was calculated in high-power fields (HPF) as follows: [10] sections with <1% positive cells were rated as 0; 1-25% positive cells as 1; 26-50% positive cells as 2; 51-75% positive cells as 3, and 76-100% positive cells as 4. The staining intensity was rated as follows: 1 for weak intensity; 2 for moderate intensity; and 3 for high intensity. Points for staining intensity and the percentage of positive cells were multiplied. Tumor specimens were classified into three groups according to overall scoring: 0-1 as negative expression, 2-4 as weak expression, and 6-12 as high expression. Total scores were expressed as: 0-4 as low and 6-12 as high. All sections were evaluated independently by two pathologists without knowledge of the identity of patients and the clinical outcome.

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Southwest Hospital, Third Military Medical University (Army Medical University). A total of 1 × 10 5 MPC1overexpressing or control cells were suspended in 50 μl PBS and subcutaneously injected into 6-week-old female NOD-SCID mice (Laboratory Animal Center, Southwest Hospital, Third Military Medical University, China). The size of resultant tumors was measured every 7 days for a month using a Vernier caliper and tumor volume was calculated as: shortest diameter 2 × longest diameter/2. The animals were sacrificed 30 days after tumor cell implantation and subcutaneous xenograft tumors were analyzed by IHC. Tumor tissue sections deparaffinized and hydrated by a series of xylene and alcohol treatment were incubated with rabbit polyclonal anti-MPC1 (Abcam), rabbit polyclonal anti-MMP7 (Cell signaling), and mouse polyclonal anti-HIF1α (Abcam) antibodies at 4 °C overnight, followed by incubation with avidin-biotin-peroxidase (DAKO).