Horseradish peroxidase hrp labeled goat anti mouse secondary antibody

Lung Sects. (5 μm) were cut from paraffin blocks into positive charged glass slides. Ordinary steps of deparaffinization and rehydration were performed via a series of graded alcohol. Heat-induced epitope retrieval was conducted. The lung sections were washed then protein blocking using bovine serum albumin (BSA) and hydrogen peroxide was applied. After that, primary antibodies (mouse monoclonal Anti-NF-κB p65 (sc-8008) were incubated with caspase-3 (sc-65497, Santa Cruz Biotechnology Inc., Dallas, TX, USA) at a dilution of 1:150 overnight at 4 °C. After that, tissue sections were incubated with horseradish-peroxidase (HRP) labeled goat anti-mouse secondary antibody (Abcam, Cambridge, UK) for 2 h then 3,3′-diaminobenzidine (DAB) Substrate detection kit (Thermo Scientific) was applied. Counterstaining was done using Mayer's hematoxylin, dehydration followed by xylene clearance and mounting in dibutylphthalate polystyrene xylene (DPX). Area % of positive reaction was quantified using digital software (Cell Sens dimensions, Olympus software). Negative control step was achieved via omitting the primary antibody incubation step.

The protein expression of FAK, ERK, and MEK was detected by the PV-9000 two-step method. The 4 μm thick sections of tissues were dewaxed, hydrated, and underwent antigen retrieval before 3% of hydrogen peroxide was added to block endogenous peroxidase activity and then incubated at 4°C overnight with the addition of primary antibodies (mouse anti-human FAK, ERK, and MEK at the ratio of 1: 500, Cell Signaling Technology, Beverly, MA, USA). After incubation for 20 min at room temperature with polymerase auxiliary agent, the solutions were treated with horseradish peroxidase (HRP) labeled goat anti-mouse secondary antibody (Abcam Inc., Cambridge, MA, USA) at room temperature for 30 min. Diaminobenzidine (DAB) (Sigma-Aldrich St. Louis, MO, USA) was used for staining. Sections were counterstained with hematoxylin and sealed. Visible pale brown or brown cytoplasm and cell membrane were considered positive cells (PBS as a negative control instead of primary antibody; adjacent normal tissues as a positive control). Four high power fields were randomly chosen. The score was obtained from the percentage of positive cells: positive tumor cells/all tumor cells > 10% (+), positive cells ≤ 10% (-) [39 (link)]. The results of the immunohistochemistry were separately scored by two individuals using the double-blind method.