Colon adenocarcinoma patient tumour tissue sections were formalin-fixed and paraffin-embeded. Deparaffinisation and hydration were performed and followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min and microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB9 antibody was used at 2 μg ml−1 in these experiments, and was incubated at 4 °C overnight. Then PV9000 2-step plus Poly-HRP anti-mouse/rabbit IgG detection system (Zhong Shan Jin Qiao, Beijing, China) was applied. The streptavidin–biotin–peroxidase method was used for detection, and diaminobenzidine was applied as substrate (ChemMate Detection Kit, DAKO, Glostrup, Denmark). Haematoxylin was used for counterstaining. Negative controls were performed by omitting the primary antibody.
Chemmate detection kit
Manufactured by Agilent Technologies
Sourced in Denmark
The ChemMate Detection Kit is a laboratory equipment product designed for the detection and identification of chemical compounds. It provides core functions for chemical analysis and identification without further interpretation or extrapolation.
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Lab products found in correlation
6 protocols using chemmate detection kit
1
Immunohistochemical Analysis of HOXB9 in Colon Adenocarcinoma
2
Histopathological Assessment of Kidney Injury
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To detect kidney injury, the fixed right kidney was dehydrated in ethanol and embedded in paraffin. Kidney tissue blocks were cut into 2-μm-thick sections and subjected to hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and Masson’s trichrome staining. For immunohistochemical analysis of kidney tissues, we used the following antibodies: mouse monoclonal against CD68 (ED1; 1:100; Abcam, Cambridge, MA, USA) and rabbit polyclonal against tumor necrosis factor-α (TNF-α; 1:200; Abcam). Next, secondary antibody was performed using HRP-conjugated polyclonal goat anti-rabbit IgG P0447 or goat anti-mouse IgG p0448 (Dako, Glostrup, Denmark) for 1 h. The sections were visualized using 3,3-diaminobenzidine (DAB; DAKO ChemMate Detection Kit) and counterstained with Mayer’s hematoxylin.
3
Orthotopic Xenograft Model for Glioblastoma
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Orthotopic implantations were employed to the in vivo study. Twenty-six NOD/SCID mice (35-day old) were intractranially injected into the brains with 1 × 105 cells (2 mm lateral and 1 mm anterior to the bregma, 3.5 mm deep) and placed into a stereotaxic frame. The survivorship curve was draw until the last mouse had died. After injected 30 days, the original tumors were photographed, and brains were collected, fixed in neutral buffered 4% PFA solution, and embedded in paraffin. Immunohistochemical (IHC) analysis and Hematoxylin and eosin (H&E) staining were employed to histopathological evaluations of the tissues by Servicebio Technology (HONG KONG, China). Briefly, the tumor sections were incubated with primary antibodies for Ars2 (1:200) and for Ki-67 (1:500), followed by detection using the ChemMate Detection kit (Dako, Denmark).Positive reaction was indicated by brown color using DAB, and the cells were counterstained with hematoxylin.
All mice were monitored and raised under the specific pathogen-free (SPF) conditions. And the welfare and experimental procedures of mice were carried out according to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technonlogy of China, 2006) and approved by the animal ethics committee, Southwest University.
All mice were monitored and raised under the specific pathogen-free (SPF) conditions. And the welfare and experimental procedures of mice were carried out according to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technonlogy of China, 2006) and approved by the animal ethics committee, Southwest University.
4
Immunohistochemistry Protocol for Tissue Samples
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Tissue sections were deparaffinized in xylene and rehydrated. Antigen retrieval was processed in pH 6.0 sodium citrate. The sections were incubated in 3% H2O2 for 30 min to quench endogenous peroxidases and then incubated with primary antibody at 4°C overnight. Then PV6001 2-step plus Poly-HRP anti-rabbit IgG detection system (Zhong Shan Jin Qiao, Beijing, China) was applied. The streptavidin-biotin-peroxidase method was used for detection. The diaminobenzidine was applied as substrate (ChemMate Detection Kit, DAKO, Glostrup, Denmark).
5
Immunohistochemical Analysis of Kindlin-1 and Kindlin-2
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All slides were formalin-fixed and paraffin-embedded. Deparaffinization and hydration were performed and followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min and microwave for antigen retrieval in 10 mmol L 1 sodium citrate buffer (pH 6.0) for 20 min. We used affinity-purified polyclonal anti-Kindlin-1 (PKU Animal Facility) antibody at 2 µg mL 1 and anti-Kindlin-2 mouse monoclonal antibody (1:1000 dilution; Millipore, USA) 2 µg mL 1 to perform these experiments. The primary antibody was used at 4°C overnight. Then PV9000 2-step plus Poly-HRP Anti-mouse/rabbit IgG Detection System (Zhong Shan Jin Qiao, China) was applied. The streptavidin-biotin-peroxidase method was used for detection and diaminobenzidine was applied for substrate (ChemMate Detection Kit, DAKO, Denmark) [19] . Hematoxylin was used for counterstaining. Negative controls were performed by omitting the use of primary antibody.
6
Immunohistochemical Analysis of Tumor Microenvironment
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All tissues used for immunohistochemistry were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were deparaffinised in xylene and rehydrated. Haematoxylin and eosin (H&E) staining was used to evaluate the overall morphology of the metastasis and the surrounding tissue. Epitope retrieval was performed in antibody-specific buffers. The following antibodies were used: CD68 (DAKO, Glostrup, Denmark), CD163 (DAKO, Glostrup, Denmark), and CD8 (DAKO, Glostrup, Denmark). Staining was performed using kits supplied by DAKO REAL Detection System (kit 5005). Antigen-specific antibodies were applied and visualized with the ChemMate detection kit (DAKO). Slides were counter-stained with H&E. Staining intensity for CD68 and CD163 was graded by board-certified dermato-pathologists (RD, EG). Macrophage density was graded in 3 levels, level 1 corresponding to lower than 30%, level 2 between 30% and 60% and level 3 to above 60% of total cell count.
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