Formalin-fixed paraffin-embedded U87 intracranial tumors were cut into 6-μm-thick sections on a microtome. Immunohistochemical staining was performed according to standard procedures. Antigen retrieval was conducted in 0.1 M citrate buffer (pH 6.0) at 95°C for 16 min and cooled at 25°C for 1 h. After blocking, slides were incubated with primary antibodies TNFAIP2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOX2 (1:200, Boster Bioengineering Co., Wuhan, China) and ki-67 (1:200, Boster Bioengineering Co., Wuhan, China). Next, according to the manufacturer’s instructions, slides were treated with the Cell & Tissue Staining Kit HRP-DAB system (R&D Systems, Minneapolis, MN, USA). The immunohistochemical staining results were evaluated by two experienced pathologists.
Cell tissue staining kit hrp dab system
Manufactured by R&D Systems
Sourced in United States
The Cell & Tissue Staining Kit HRP-DAB system is a laboratory equipment product designed for the detection and visualization of target proteins in cells and tissues. The kit utilizes the horseradish peroxidase (HRP) enzyme and the chromogenic substrate 3,3'-diaminobenzidine (DAB) to produce a brown-colored reaction product, allowing for the identification and localization of the target protein within the sample.
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5 protocols using cell tissue staining kit hrp dab system
1
Immunohistochemical Analysis of U87 Glioma
2
Immunohistochemical Evaluation of U87 Tumors
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Formalin-fixed paraffin-embedded U87 tumors were cut with a microtome into 6-μm sections. Antigen retrieval was performed in 10 mM sodium citrate buffer of pH 6 for 16 min at 96–98 °C. Slides were incubated with primary antibodies against Ki-67 (Boster Bioengineering Co., Wuhan, China), and with antibodies against MMP2 and TIMP3 (Abcam). Sections were subsequently incubated with the Cell & Tissue Staining Kit HRP-DAB system (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Immunostaining was performed with known positive and negative tumor controls and were blindly evaluated by a pathologist.
3
Immunohistochemical Analysis of MMP2
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The 8505C tumors were formalin-fixed, embedded in paraffin, and cut into 6-μm sections. Antigen retrieval was conducted in 10 mM sodium citrate buffer (pH 6) at 96–98°C for 16 min. After that, the sections were incubated with primary antibodies against MMP2 (Abcam). Subsequently, according to the instructions, sections were incubated with the Cell & Tissue Staining Kit HRP-DAB system (R&D Systems, Minneapolis, MN). Immunostaining was conducted with known positive and negative tumor controls and were blindly assessed by a pathologist.
4
Immunohistochemical Detection of Osteocalcin
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The deparaffinized sections were washed with PBS, treated with osteocalcin (OC; 1:10000, mouse anti-pig osteocalcin monoclonal IgG, ab13418, Abcam, Cambridge, UK) and incubated over night at 4 °C (1:10000, mouse anti-pig osteocalcin monoclonal IgG, ab13418, Abcam, Cambridge, UK). The primary antibody was followed by incubation with the secondary antibody (1:200, horse anti-mouse IgG, BA-2001, Vector Laboratories, Burlingame, USA) at room temperature for 1 hr. A final incubation was performed using the tertiary complex streptavidin peroxidase (Pierce™ High Sensitivity Streptavidin-HRP, Catalogue no: 21130, ThermoFisher, Germany) for an additional 30 minutes. The reaction was visualized using Cell & Tissue Staining Kit (HRP-DAB system, R & D Systems, McCinley, Canada) according to manufacturer’s recommendations.
5
Quantifying Macrophage Infiltration in Bone
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Tibiae were fixed in 4% paraformaldehyde/PBS (4°C, 24hr), decalcified in 14% EDTA for 2 weeks, and embedded in paraffin. Immunohistochemical staining was performed, using the Cell & Tissue Staining Kit (HRP-DAB system; R&D systems) and rat monoclonal anti-mouse F4/80 (1:100, Abcam ab6640, Cambridge, UK). Percentages of F4/80+ cells were quantified using NIS Elements software (Nikon). The mean area positive for F4/80+ cells was calculated for each tissue within 4 different areas of the tissue.
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