Envision system anti rabbit

Paraffin tissue sections were quenched for 10 min in aqueous 3% hydrogen peroxide. Epitopes were retrieved using an in-house glycan retrieval solution, in a Biocare Medical Decloaking Chamber. The primary antibody applied to the sections was SARS-CoV-2 (2019-nCoV) Nucleocapsid, Rabbit MAb (Sino Biological Inc. #40143-R019, Beijing, China). It was used at a 1:6000 dilution for thirty minutes. The sections were then visualized using a horse radish peroxidase-labelled polymer, Envision^® + system (anti-rabbit) (Dako, Agilent, Santa Clara, CA, USA), and reacted with the chromogen diaminobenzidine (DAB). The sections were then counter-stained with Gill’s hematoxylin.

Specimens from primary tumor or metastases were obtained from patients undergoing surgery within four weeks of the PET/CT. The specimens were placed in formalin and paraffin embedded within 24 h. The samples were cut in sections of 4 µM and dewaxed through xylene to tap water. For antigen retrieval the sections were treated with proteinase K for 5 min. This was followed by a blocking step with Peroxidase-Blocking Solution (Agilent, S2023) and pre-incubation in 2% BSA for 10 min. For visualizing the intensity and distribution of integrin, α_vβ₃ sections were incubated with primary antibody (Absolute Antibodies, Ab00890-23.0) in a 1:50 dilution in 2% BSA overnight at 40 °C [19 (link)].
For visualization, the sections were incubated with Envision+ system Anti-Rabbit (Agilent, K4003) for 45 min followed by incubation with DAB+ system (Agilent, K3468) for 10 min. Counterstaining was performed with Mayer’s Hematoxylin. The sections were visually evaluated regarding α_vβ₃ intensity.