Paraffin-embed tissues including HCC tumor tissues and normal liver tissues were cut into 3 µm thickness. The tissue slides were de-paraffinized using xylene and hydrated in a decreasing gradient of ethanol before incubating them with 3% hydrogen peroxide to block endogenous peroxidase activity for 20 min. Antigen retrieval was carried out using target retrieval solution (Dako, Carpinteria, CA, USA) followed by blocking non-specific binding with biotin blocking system (Dako) for 10 min. Thereafter, the slides were incubated with anti-ASS antibody (1:100, BD Biosciences) overnight, and biotinylated anti-mouse antibody (Dako) for 15 min, and streptavidin conjugated peroxidase for another 15 min. The chromogen development is performed using DAB Substrate-Chromogen (Dako). The slides were counterstained with hematoxylin, dehydrated with ethanol and mounted with Cytoseal-60. ASS expression on tissues slides were scored by a pathologist based on intensity of ASS positive cells in more than 4 different areas using the microscope with high power objective (100×).
Biotinylated anti mouse antibody
Manufactured by Agilent Technologies
The Biotinylated anti-mouse antibody is a laboratory reagent used to detect and quantify mouse proteins in various research applications. It is a secondary antibody conjugated with biotin, a small molecule that can bind to streptavidin or avidin, enabling signal amplification and detection techniques.
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Lab products found in correlation
Dab substrate chromogen, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Biotin blocking system, by Agilent Technologies (1 mentions)
Streptavidin cy3, by Merck Group (1 mentions)
Streptavidin peroxidase, by Agilent Technologies (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
2 protocols using biotinylated anti mouse antibody
1
Immunohistochemical Analysis of ASS in HCC
2
Histochemical Analysis of Muscle Transplants
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Sections were stained with H&E for standard analysis. For β-gal histochemical detection in muscles transplanted with LacZ+ cells, sections were fixed 3 min in 0.25% glutaraldehyde, rinsed with PBS, and incubated 24 hr at room temperature in 0.4 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) (Boehringer Mannheim) with 1 mM MgCl2, 3 mM Ke3F(CN)6,·3 mM Ke4F(CN)6, and·3H20 in PBS. To depict acute rejection,35 (link) we immunodetected CD8+ lymphocytes using a mouse anti-human/macaque CD8 monoclonal antibody (BD Biosciences), followed by a 30-min incubation with a biotinylated anti-mouse antibody (1/150, Dako) and either a 30-min incubation with streptavidin-Cy3 (1/700, Sigma) or a 30-min incubation in streptavidin-peroxidase (1/200, Dako). Peroxidase activity was revealed with 3,3′ diaminobenzidine (0.25 mg/mL) and 0.03% hydrogen peroxide. Reagents for immunodetection were diluted in PBS with 1% FBS. Stained sections were analyzed using an Axiophot microscope with epifluorescence and bright-field optics (Zeiss). To estimate the density of CD8+ cells, we counted the number of CD8+ cells per muscle section and the area of the section was measured using ImageJ software.
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