Peroxidase conjugated biotin streptavidin complex

Thawed samples were fixed in 4% formalin and embedded in paraffin for histopathological analysis. Samples were deparaffinized with xylol and then sliced into 4-µm sections. Sections were rehydrated using a graded ethanol series. A heat-induced epitope protocol was used for antigen-retrieval (95°C for 40 min). Samples were incubated in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase. Samples were blocked with protein serum (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA, USA) and then incubated (overnight at 4 °C) with polyclonal rabbit anti-human EVA1A antibody at 1:1000 (Abcam, Shanghai, China). After washing three times in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 7.6), sections were incubated with secondary antibody for 20 min at room temperature. Peroxidase-conjugated biotin-streptavidin complex (Dako, Glostrup, Denmark) was then applied to the sections for 20 min. Sections were visualized with 3, 3′-diaminobenzidine and counterstained with hematoxylin. The negative control used nonimmune serum instead of primary antibody.

Immunohistochemical staining was performed using a method described previously (22 (link)). Briefly, thawed samples were fixed in 4% formalin and embedded in paraffin for histopathological analysis. Samples were deparaffinized with xylol and then sliced into 4 µm sections. Sections were rehydrated using a graded ethanol series. A heat-induced epitope protocol was used for antigen-retrieval (95°C for 40 min). Samples were incubated in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase. Samples were blocked with protein serum (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA, USA) and then incubated (overnight at 4°C) with polyclonal rabbit anti-human CCNG2 or Ki67 antibody at 1:1,000 (MBL International Corporation, Nagoya, Japan). After washing three times in TBST (150 mM NaCl, 10 mM Tris–HCl, pH 7.6), sections were incubated with secondary antibody for 20 min at room temperature. Peroxidase-conjugated biotin-streptavidin complex (Dako, Glostrup, Denmark) was then applied to the sections for 20 min. Sections were visualized with 3, 3′-diaminobenzidine and counterstained with hematoxylin. The negative control used nonimmune serum instead of primary antibody.

The xylol-deparaffinized samples were sectioned into 4 µm slices and then rehydrated using a graded series of ethanol. Antigen retrieval was performed using a heat-induced epitope protocol at 95 °C for 40 min. To block endogenous peroxidases, the sections were incubated in methanol containing 0.3% hydrogen peroxide. Samples were blocked with protein serum using a Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA), then incubated overnight at 4 °C with anti-α-SMA, anti-E-cadherin, and anti-Collagen IV antibody (CST, MA, USA) at a dilution of 1:1000. After washing with TBST, the sections were exposed to a secondary antibody for 20 min at room temperature, followed by treatment with a peroxidase-conjugated biotin-streptavidin complex (Dako, Glostrup, Denmark) for 20 min. Visualisation was achieved using 3,3'-diaminobenzidine, and subsequent counterstaining was performed with hematoxylin. For the negative control, nonimmune serum was utilised in place of the primary antibody.