Tissue sections that were embedded in paraffin slides were deparaffinized in xylene and then rehydrated in graded alcohol solutions. The endogenous peroxidase activity was inhibited by incubating the slide in 3% (v/v) hydrogen peroxide, and the antigenicity was recovered with a 0.01 mol l−1 sodium citrate buffer (pH 6.0, Cat. No. AR0024, Boster Biological Technology, Wuhan, China). The samples were incubated overnight at 4°C with antibodies for UCHL1 (dilution: 1:100, Cat. No. 13179, Cell Signaling Technology, Danvers, MA, USA), SOX9 (dilution: 1:100, Cat. No. AB5535, Millipore, Burlington, MA, USA), and α-SMA (dilution: 1:100, Cat. No. ARG52485, Arigo, Hsinchu, Taiwan, China); washed with PBS; and then incubated with Cy3-conjugated goat anti-rabbit (dilution: 1:100, Cat. No. CW0159S, CWBIO, Beijing, China), Cy3-conjugated goat anti-mouse (dilution: 1:100, Cat. No. CW0145, CWBIO), and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (dilution: 1:100, Cat. No. CW0114S, CWBIO) at room temperature for 30 min. Subsequently, the sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 10 μg ml−1, Cat. No. C0065, Solarbio, Beijing, China). Immunofluorescence signals were detected by fluorescence microscopy (IX71, OLYMPUS).
Ar0024
Manufactured by Boster Bio
The AR0024 is a laboratory instrument designed for scientific research and analysis. It is a high-performance spectrophotometer capable of measuring the absorbance of light in a sample across a wide range of wavelengths. This device can be used to quantify the concentration of various analytes in a sample through the principle of spectrophotometry.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (2 mentions)
α sma, by Merck Group (1 mentions)
Hematoxylin, by Maclin Biochemical Technology (1 mentions)
Ab128874, by Abcam (1 mentions)
Bx53 optical microscope, by Olympus (1 mentions)
Antibody diluent, by Beyotime (1 mentions)
G1052, by Wuhan Servicebio Technology (1 mentions)
Gfap ga5 mouse, by Cell Signaling Technology (1 mentions)
Sp rabbit and mouse hrp kit, by CWBIO (1 mentions)
Ab32572, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
Sc 28320, by Santa Cruz Biotechnology (1 mentions)
5 protocols using ar0024
1
Immunofluorescence Staining of Tissue Sections
2
Immunohistochemistry Analysis of BRD4 in Mouse Tumor Tissue
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After being fixed on a glass slide, the mice tumor tissue slices were soaked in citrate antigen repair solution (AR0024, Boster, Wuhan, China) and heated in a microwave oven for 8 min. Then the tissue slides were cooled naturally at room temperature and incubated with BRD4 (1:400, ab128874, Abcam) at 4 °C overnight. The next day, the tissue slides were incubated with the secondary antibody (ab205718, Abcam) for 2 h and stained with the DAB buffer for 35 min. After 2 min of washing under tap water, hematoxylin (H810910, Macklin) was added to stain the nuclei of the cells for 4 min. Finally, after the tissue slides were sealed, the images of the tissue slices were recorded using a BX53 optical microscope (Olympus, Japan).
3
Histopathological Analysis of TBSI Animal Model
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To verify the success of the TBSI animal model, histopathological analysis was performed on paraffin-embedded brain tissue (8 rats per group). Bain stem tissues were fixed in 4% paraformaldehyde for 24 h. The cut surface was treated with a gradient of alcohol, xylene and paraffin, and the wax blocks were sectioned serially (5 μm) for hematoxylin and eosin (H&E) staining, silver staining (G1052, Servicebio, Wuhan, China) and immunohistochemistry (IHC) for GFAP. IHC was performed according to the protocol recommended in the instructions. Sections were dewaxed with xylene and hydrated in a series of ethanol solutions, followed by high temperature antigen repair by microwave in 0.01 M sodium citrate antigen repair solution (pH 6.0, AR0024, BOSTER, Wuhan, China) for 20 min. Subsequent blocking was performed according to the SP Rabbit and Mouse HRP Kit (CW2069, CWBIO, Taizhou, China) and after washing, primary antibody [GFAP (GA5) Mouse, #3670, Cell Signaling Technology, Boston, MA, United States] was diluted 1:200 with Antibody Diluent (P0262, Beyotime, Shanghai, China) and incubated overnight at 4°C. DAB color development was performed according to the kit the next day and the nuclei were stained with hematoxylin followed by xylene transparency and finally sealed with neutral gum.
4
Immunohistochemical Analysis of β-catenin
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The airway tissues were fixed with 4% paraformaldehyde for 24 h at room temperature. Then the tissues were washed overnight under tap water and dehydrated with gradient ethanol, which was prepared with different volumes of 100% ethanol and double distilled water. Subsequently, the tissues were paraffin-embedded, sectioned, and dewaxed. After soaking the tissues in 3% hydrogen peroxide (C804187, Macklin) for 25 min and in citrate antigen repair solution (AR0024, Boster, Wuhan, China), the tissue slices were heated in a microwave oven (BE01, Galanz, Guangdong, Shanghai) for 8 min. Then the tissue slices were naturally cooled at room temperature and then incubated with β-catenin antibody (1:250, ab32572, Abcam) overnight at 4°C. The second day, the slices were further incubated with goat-anti-rabbit IgG (1:30000, ab205718, Abcam) antibody for 1 min, followed by incubation with the DBA reagent (SFQ004, 4A Biotech, Beijing, China) for 30 min. After being washed for 1 min with tap water, the tissues were stained with hematoxylin for 4 min. Then the tissues were soaked in gradient ethanol again for dehydration and made transparent by rinsing in xylene. Finally, the tissue slices were sealed with neutral gum and the image of each section was collected under a DMLA fully automatic microscope (Leica, Solms, Germany) at a magnification of 100 ×.
5
Immunohistochemical Analysis of Flotillin-2
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Representative areas of tumor cores in the paraffin embedded tissue blocks were selected according to hematoxylin-eosin (H&E) staining, and then transferred into a recipient master. Antigen retrieval was performed by immersing the sections in citrate buffer (pH 6.0; cat. no. AR0024; Boster Biological Technology) and heating in a microwave for 5 min. The sections were subsequently treated with 3% hydrogen peroxide for 30 min at room temperature to quench endogenous peroxidase activity, followed by 10% normal goat serum for 30 min at 37°C. Sections were incubated with mouse monoclonal antibody against human flotillin-2 (cat. no. sc-28320; 1:200; Santa Cruz Biotechnology, Inc.) overnight at 4°C followed by incubation with anti-mouse secondary antibody for 30 min at 37°C. The sections were subsequently stained with DAB (Beyotime), and the staining was evaluated by two independent observers.
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