Secondary anti rabbit igg

Immunohistochemistry was performed on fresh frozen slides (tumor/normal pairs) using standard IHC protocol. A fresh frozen sample of pancreatic adenocarcinoma was selected as the positive control. IHC staining was performed on 5 mm-thick sections of fresh frozen samples with purified rabbit anti human IGF2BP3 polyclonal antibody (Protein Tech Group, INC). The slides were incubated in H2O2 solution for 10 minutes at room temperature to block the endogenous peroxidase activity. Antigen retrieval was performed by steamer heating in 10mmol/L citrate buffer (pH 6.0). After epitope recovery, slides were incubated with primary antibody IGF2BP3 at 1:100 dilutions overnight at 4°C temperatures. Slides were washed and incubated with secondary anti-rabbit IgG (Vector laboratories, CA) at 1:200 dilution and tertiary streptavidin-peroxidase conjugate (ABC complex, Vector laboratories, CA). Slides were treated with chromogen “diaminobenzedine” for antigen detection. Counterstaining was performed with hematoxylin, dehydrated, cleared in xylene, and mounted on coated slides.

Slide sections of kidney and tumor tissues were treated with xylene and ethanol and then boiled in sodium citrate buffer for 20 min. Tissue sections were treated with 5% H₂O₂ for 15 min to inactivate endogenous peroxidase. Kidney slides were reacted with KIM-1 (1:1000), SBP1 (1:1000), and NGAL (1:1000) antibodies. Slides were reacted with Ki-67 (1:1000) at 4 °C for 24 h. The secondary anti-rabbit IgG (VECTOR laboratories, Burlingame, CA, USA) was allowed to react at room temperature for 30 min. HRP-streptavidin reagent (VECTOR laboratories) was reacted at room temperature for 30 min. Following staining by DAB (Dako, Agilent, Santa Clara, CA, USA) and hematoxylin (Dako), the slides were fixed on a cover glass using a mounting solution after ethanol and xylene treatment. Slides were observed at 200× magnification by a confocal K1-fluo microscope (Nanoscope Systems, Daejeon, Korea).