Dab peroxidase substrate solution

FFPE (formalin fixed paraffin embedded) liver tumor tissue sections obtained from patients were subjected to immunostaining using AS1411 aptamer. The tissue sections were deparaffinized twice in xylene for 15 min each, washed with series of 100%, 95%, 80% and 70% for 1 min each and rinsed with distilled water. Tissue sections were then incubated in 10 mM sodium citrate buffer (pH 6.2) at 95°C for 30 min, samples were then microwaved for 2 min and allowed to cool at room temperature. After being washed twice in PBS for 5 min each, tissue sections were incubated in 3% H₂O₂ in PBS for 30 min then blocked with biotin-blocking solution (Vector, Laboratories). Tissue sections were then probed with 200 nM AS1411-Biotin for 1h at room temperature and washed 3 times in 0.5% PBST and incubated with HRP-conjugated streptavidin solution (Dako) for 30 min at room temperature. Finally, tissue sections were treated with DAB peroxidase substrate solution (Dako) until color developed (approximately 5 min). Stained tissue sections were examined and pictures were taken by a light microscope.

Samples for histological analysis were fixed in 4%PFA overnight at 4°C immediately after collection from the animals. For immunohistochemistry analysis, 5 μm sections were de-waxed and re-hydrated through an ethanol scale, heated in citrate solution (BioGenex #HK086-9K) in a water bath at 99°C for 30 minutes for antigen unmasking, washed once in water and treated with 3% H₂O₂ for quenching of endogenous peroxidases. Slides were incubated with antibody against Ki-67 (#M7249 Dako, 1:500) or Cleaved Caspase-3 (#9661 Cell Signaling, 1:200), in a blocking solution (2% BSA, 2% goat serum, 0.02% Tween20, in TBS 1x) for 3h at RT. After primary incubation, slides were washed twice with TBS 1x, and incubate with the secondary antibody (DAKO Cytomation Envision System Labelled Polymer-HRP) for 45 minutes. After 2 washes with TBS, the signal was revealed with a DAB peroxidase substrate solution (DAKO) for 2-10 minutes. Slides were finally counterstained with hematoxylin, de-hydrated through alcoholic scale and mounted with Eukitt (Bio-Optica).

Formalin fixed paraffin tissue sections (4 μm) were used with an indirect peroxidase labeling technique (Envision Plus, DAKO, Australia). Following deparaffinization and rehydration, endogenous peroxidase activity was blocked with 3 % H₂O₂ and non-specific binding inhibited with 10 % normal goat serum (01–6201 Zymed Laboratories, USA). Heat induced epitope retrieval was used. Antigens were visualised by immunohistochemical staining using their respective antibodies diluted as follows: (CD34 1:500, AbD serotec MCA18256; Ki-67 1:100, Thermoscientific, RM-9106-S1; caspase 3 1:800, R&D AF835) and EMT markers (ZEB1 1:200, Santa Cruz sc-25388; Vimentin 1:300, Santa Cruz sc-5565; E-cadherin 1:500 Sana Cruz sc-7870; b-catenin 1:300, Santa Cruz sc-7199) Negative controls were stained by the corresponding isotype antibodies. Following primary antibody treatment, sections were incubated with a horseradish peroxidase labelled polymer secondary antibody. The antigen-antibody complex was visualized by diaminobenzene (DAB) Peroxidase Substrate Solution (DAKO, Australia). Each treatment group consisted of 5–8 animals. A minimum of ten tumors were assessed for each treatment group and from 3 to 15 images per tumor, depending on tumor size, were captured for analysis.