Paraffin-embedded sections were 5-μm thick sections, deparaffinized with xylene and ethanol, and then heated in 0.1 mol/L citrate buffer (pH 6.0) by microwaving for 15 min. After being cooled at room temperature, the sections were incubated in 3% hydrogen peroxide for 20 min to block endogenous peroxidase. Then, the sections were blocked with 10% (v/v) BSA (Sangon, Shanghai, China) to inhibit non-specific binding, followed by incubation with primary antibody for ACTN1 (Abcam, ab50599) at 4 °C overnight with a dilution at 1:200. After washing with PBS for three times, sections were incubated with secondary reagent. All the slides were labeled with DAB substrate liquid (Cell signaling Technology, USA) and counterstained by hematoxylin, and photographed with a microscope (Carl Zeiss, USA). Scoring was conducted according to the intensity of positive staining and the proportion of stained tumor cells (score 0: 0–5%, 1: 6–30%, 2: 31–70%, and 3: > 71%. Scoring was evaluated by two independent investigators who were blinded to the clinical information. Disagreements were resolved by consensus.
Dab substrate liquid
Manufactured by Cell Signaling Technology
Sourced in United States
DAB substrate liquid is a ready-to-use solution for the visualization of peroxidase-based immunohistochemistry and immunocytochemistry. The core function of this product is to provide a chromogenic substrate that reacts with the peroxidase enzyme, resulting in the deposition of a brown-colored precipitate at the site of the target antigen.
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Lab products found in correlation
2 protocols using dab substrate liquid
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Immunohistochemical Analysis of ACTN1
2
Immunohistochemical Evaluation of ACTN1
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Para n-embedded sections were 5-μm thick sections, depara nized with xylene and ethanol, and then heated in 0.1 mol/L citrate buffer (pH 6.0) by microwaving for 15 min. After being cooled at room temperature, the sections were incubated in 3% hydrogen peroxide for 20 minutes to block endogenous peroxidase. Then, the sections were blocked with 10% (v/v) BSA (Sangon, Shanghai, China) to inhibit non-speci c binding, followed by incubation with primary antibody for ACTN1 (Abcam, ab50599) at 4 °C overnight with a dilution at 1:200. After washing with PBS for three times, sections were incubated with secondary reagent. All the slides were labeled with DAB substrate liquid (Cell signaling Technology, USA) and counterstained by hematoxylin, and photographed with a microscope (Carl Zeiss, USA). Scoring was conducted according to the intensity of positive staining and the proportion of stained tumor cells (score 0: 0-5%, 1: 6-30%, 2: 31-70%, and 3: > 71%. Scoring was evaluated by two independent investigators who were blinded to the clinical information. Disagreements were resolved by consensus.
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