Ab122396

The CCDC34 and VEGF levels and the presence of CD34 were assessed by immunohistochemistry. The experimental method is a 2-step method. First, sections were first dewaxed in phosphate-buffered saline (PBS), then incubated in 10 mmol/l pH 6.0 citrate buffer, heated in a microwave oven, and incubated in 3% hydrogen peroxide for 15 min at room temperature. Second, sections were incubated with primary antibodies (CCDC34 antibody [ab122396; Abcam Inc, Shanghai, People’s Republic of China], VEGF antibody [ab2349; Abcam Inc, Shanghai, People’s Republic of China], and CD34 antibody [ab81289; Abcam Inc, Shanghai, People’s Republic of China]), all at a dilution of 1: 100 at 4°C overnight. We removed the humidified chamber with sides from the freezer, equilibrated it to room temperature for 45 min, and then absorbed the surplus primary antibody. the sides were incubated at room temperature for 30 min with the appropriate secondary antibody (Envision+HRP, Rabbit, DAKO). Finally, sections were colored through reacting with 3.3-diaminobenzidine. Hematoxylin was applied as counterstain, and negative-control sections were processed with PBS.

Immunohistochemical staining was performed according to the manufacturer’s protocol, using the primary antibody to CCDC34 antibody (ab122396) (Abcam, Cambridge, UK) at a dilution of 1: 500. The results were determined by light microscopy, with the immunostaining of each tissue section independently assessed by two histopathologists, as previously reported [8 (link)]. A visual immunoreactive score (IRS) was used for each case, ranging from 0–12, which was generated by staining intensity (SI) × number of stained cells (PP). The detailed scoring criteria for the SI (score, 0–3): 0 was negative, 1 was weakly positive, 2 was moderately positive and 3 was strongly positive. The detailed scoring criteria for the PP (score, 0–4): 0 was negative, 1 was 1–25%, 2 was 26–50%, 3 was 51–75%, 4 was 76–100%. If the IRS score was >4, the CCDC34 expression was defined as high; and if the IRS score was ≤4, the CCDC34 expression was defined as high.

Immunohistochemistry was performed to examine the expression levels of CCDC34 (ab122396, Abcam, Inc.), VEGF (ab2349, Abcam, Inc.) and CD31 (ab28364, Abcam, Inc.) according to the manufacturer’s instructions. The working concentration of each antibody was at a dilution of 1: 100. Immunohistochemical scores were calculated based on a previously reported method [5 (link)]: “+” score for ≥10% of tumor cells positive; “−” score when less than 10% of tumor cells or no visible staining was detected.