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Dab reagent

Manufactured by Solarbio

The DAB reagent is a chromogenic substrate used in immunohistochemistry and immunocytochemistry techniques. It produces a brown-colored reaction product at the site of the target antigen, allowing for the visualization and localization of the antigen within the sample.

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3 protocols using dab reagent

1

Evaluating Lung Fibrotic Phenotype via IHC

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IHC was performed to evaluate the fibrotic phenotype of lung tissues. After deparaffinization and hydration, the slices were heated with the antigen retrieval solution for 10 min. Then, the slices were penetrated with PBS for 5 min followed with H2O2 treatment for about 15 min, and then blocked with goat serum (SL038, Solarbio) for 60 min at RT to eliminate the activity of endogenous peroxidase. The slices were incubated overnight at 4°C with antibodies against the following proteins: α-SMA (1:200, AF1032, Affinity) and fibroblast-specific protein 1 (FSP-1, 1: 200, A19109, Abclonal). The next day, the slices were incubated with HRP-labeled secondary antibody (1:500, #31460, ThermoFisher, USA) at 37°C for 60 min. After developed with DAB reagent (DA1010, Solarbio), the slices were then counterstained with hematoxylin (H8070, Solarbio). Finally, the slides were mounted and observed under a microscope.
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2

Apoptosis Detection in Fibroblasts

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TUNEL assay was performed to detect apoptosis of fibroblasts. Cells cultured on glass slides were fixed by 4% paraformaldehyde (Sinopharm) for 15 min, permeated with 0.1% TritonX-100 for 8 min, blocked with 3% H2O2 (Sinopharm) for 10 min, and incubated with TUNEL reaction mixture (Roche, Basel, Switzerland) at 37°C for 60 min. After rinsing with PBS, cells were incubated with Converter-POD (Roche) at 37°C for 30 min, counterstained with DAB reagent (Solarbio), and counterstained with hematoxylin (Solarbio) for 3 min. Then the cells were treated with 1% hydrochloric acid alcohol, rinsed with water for 20 min, and mounted with glycerine ethanol. Thereafter, the cells were observed and photographed with a microscope (Olympus, Tokyo, Japan) at 400× magnification.
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3

Immunohistochemical Analysis of USP39 Protein

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The cells and tissues were fixed with a fixator, then embedded with paraffin, and finally sectioned in paraffin blocks. Paraffin sections were cleaned and dewaxed with ethanol solutions of different concentrations. Antigen repair solution was used to repair the antigen, and PBS was washed 3 times. Polyclonal rabbit anti-USP39 antibody (Abcam, US) was added and incubated overnight at 4°C, then washed with PBS solution. HRP-labeled secondary antibody (Abcam, US) was added and incubated at 4°C for 30 min: DAB dyeing with DAB reagent (Solarbio Life Science, Beijing), microscope observation, and photography.
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