VEGF and HIF-1α immunohistochemistry

Prior to PRCT, a colposcopic biopsy of the cervical tumor was performed. A radical hysterectomy was performed 2 weeks post PRCT, and the resected tumor lesions were used in the immunohistochemical assays. Immunohistochemistry reagents were all purchased from Maixin Biotech Co., Ltd. (Fuzhou, Fujian, People’s Republic of China). The streptavidin–peroxidase–biotin method was utilized in accordance with the manufacturer’s instructions. Tissue sections were first incubated with mouse monoclonal anti-VEGF and anti-HIF antibodies at a dilution of 1:50, respectively. Then, a secondary antibody (Supervision_Universal [Anti-Mouse/Rabbit] Detection Reagent) and horseradish peroxidase were applied.

Positive VEGF staining was indicated by the presence of brownish–yellow granules in the cytoplasm, while HIF-1α staining was mainly present in the cytoplasm with very little expression in the nucleus (Figures 1 and ​and2).2). Staining was evaluated with a nomogram as reported by Qiu et al, which incorporates both the proportion of positive staining cells and the staining intensity.7 Briefly, the percentage of positive cells was scored as 0 (0% positive cells), 1 (1%–25% positive cells), 2 (26%–50% positive cells), and 3 (>50% positive cells). The intensity of immunostaining was scored as 0 (negative), 1 (weak), 2 (intermediate), and 3 (strong). The intensity score (0–3) was multiplied by the percentage score (0–3) and a final score was assigned 0–1 (negative), 2–3 (+, weak expression), 4–5 (++, moderate expression), and 6 (+++, strong expression). The results of the VEGF and HIF-1α assays were determined by two professional pathologists using a double-blind method. Disagreement was addressed and reviewed by a senior physician.

Immunohistochemical staining of VEGF in LACC lesion.

Notes: Original magnification ×400. (A) Negative, (B) weak expression, (C) moderate expression, (D) strong expression.

Abbreviations: LACC, locally advanced cervical cancer; VEGF, vascular endothelial growth factor.

Immunohistochemical staining of HIF-1α in LACC lesion.

Notes: Original magnification ×400. (A) Negative, (B) weak expression, (C) moderate expression, (D) strong expression.

Abbreviations: HIF-1α, hypoxia inducible factor-1α; LACC, locally advanced cervical cancer.