3 3 diaminobenzine dab

Paraffin-embedded rat kidney slides were deparaffined, rehydrated, and immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. All sections were heated in Tris-EDTA buffer (pH 9.0, Boster, Wuhan, China), blocked with 5% blocking buffer for 30min at 37°C, incubated with primary antibodies against fibronectin (FN) (1:100, Abcam Cat# ab2413, Cambridge, England), type I collagen (Col-I) (1:100, Abcam Cat# ab254113, Cambridge, England) at 4°C overnight, incubated with species-specific secondary antibody (SV0004, Boster, Wuhan, China), developed with 3,3′-diaminobenzine (DAB, Invitrogen, California, United States) and counterstained with hematoxylin. The integrated optical density (iod) values of the positive staining areas were measured by ImagePro Plus 6.0 software (Media Cybernetics, CA, United States).

Paraffin-embedded human specimens and xenografts were deparaffined and rehydrated. All sections were heated in Tris-EDTA buffer (pH 9.0), treated with hydrogen peroxide, and incubated with primary antibodies against AGO2, Ki-67 (Abcam-ab92742), cleaved Caspase-3 (Abcam-ab32042) and E-cadherin (Abcam-ab76319) respectively. Then sections were incubated with species-specific secondary antibody (SV0004, Boster, Wuhan, China), developed with 3,3’-diaminobenzine (DAB, Invitrogen) and counterstained with hematoxylin.
IHC staining and scoring were determined by two clinical pathologists respectively according to the proportion of positively stained cells and staining intensity. The positively stained cells was scored: 1 (<10%), 2 (10-30%), 3 (30-60%), and 4 (>60%). The staining intensity was graded: 1 (light yellow), 2 (yellow to brown), 3 (brown), and 4 (deep brown). Staining index (SI) was determined by positive staining cells × staining intensity. The expression level of AGO2 was scored according to the SI. Score 1 and 2 were considered as negative, rest of the scores as positive.

Paraffin-embedded rat kidney slides were deparaffined, rehydrated, and immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity. All sections were heated in Tris-EDTA buffer (pH 9.0, Boster, Wuhan, China), blocked with 5% blocking buffer for 30 min at 37 °C, incubated with primary antibodies against Tumor necrosis factor (TNF-α) (1:100, Abcam Cat# ab6671, Cambridge, England), IL-6 (1:100, Abcam Cat# ab9324, Cambridge, England) at 4 °C overnight, incubated with species-specific secondary antibody (SV0004, Boster, Wuhan, China), developed with 3,3′-diaminobenzine (DAB, Invitrogen, California, USA) and counterstained with hematoxylin. The integrated optical density (IOD) values of the positive staining areas were measured by ImagePro Plus 6.0 software (Media Cybernetics, CA, USA).