Gtvisiontmiii detection system mo rb

Four μm thick paraffin sections were stained for mTOR, p-mTOR, S6 and p-S6 expression. Slices were deparaffinized and hydrated. Antigen retrieval was achieved by pressure cooking in 10 mM sodium citrate buffer, pH 6.0 for 10 mins, and incubating with primary antibody overnight (1:100 for mTOR, p-mTOR and S6, 1:400 for p-S6) at 4°C. Detection took place by GT Vision^TM III Detection System/Mo Rb (GeneTech, Shanghai, China) and colorimetric detection with 3,3′-diaminobenzidine. Afterwards, the slides were dehydrated and mounted with coverslips.

In the immunocytochemical assay, the slides were rehydrated and immersed in 3% hydrogen peroxide solution for 15 min; pretreated by microwave for 25 min in 0.01 mol l⁻¹ citrate buffer, pH 6.0, at 95 °C; and cooled for 60 min at room temperature. In between each incubation step, the sections were washed with PBS, pH 7.4. The slides were blocked by 10% normal goat serum for 30 min at 37 °C, washed, and then incubated overnight at 4 °C with diluted antibody against each protein studied. After washing with PBS, the slides were visualized using GTVisionTMIII Detection System/Mo&Rb (GeneTech, GK500710) following the manufacturer's instructions. IHC staining for POH1 and E2F1 was cored according to the intensity (1: low staining; 2: moderate staining; 3: high staining; 4: extremely high staining), the percentage of positive cells (1: 0–25%; 2: 26–50%; 3: 51–75%; 4: 76–100%), and the location of staining (1: no nuclear staining or the intensity of nuclear staining was weaker than that of cytoplasm; 2: the intensity of nuclear staining was equal to that of cytoplasm; 3: the intensity of nuclear staining was higher than that of cytoplasm). The overall score=intensity score × percentage score × location score.

The tissue microarrays and slides were deparaffinized, rehydrated, immersed in 3% hydrogen peroxide solution for 15 min, heated in citrate buffer (pH 6.0) for 25 min at 95 °C, and cooled for at least 60 min at room temperature. Between each incubation step, the slides were washed three times with PBS (pH 7.4). After blocked with 10% normal goat serum for 30 min at 37 °C and washed, the slides were incubated overnight at 4 °C with primary antibodies against target proteins and visualized using the PV-9000 Polymer Detection System (GBI, USA) following the manufacturer’s instructions or GTVisionTMIII Detection System/Mo&Rb (GeneTech, GK500710). After washing with PBS, the slides were counterstained with hematoxylin.

Tissue microarrays were constructed using paired CRC and NCT tissues. IHC staining was performed on 4-mm sections of paraffin-embedded tissue samples to detect the expression levels of CREB1 protein. In brief, the slides were incubated in CREB1 antibody diluted to 1:150 at 4 °C overnight. The subsequent steps were performed using the GTVisionTM III Detection System/Mo&Rb (Gene Tech, China).