The immunohistochemical assay for glial fibrillary acidic protein (GFAP) was conducted for 3 animals of each experimental group. To prevent antigen degradation, sections were stored at 4 °C prior to assay. Briefly, brains were fixed in 4% (w/v) p-formaldehyde for 48 h, embedded in paraffin and sliced into 5-micrometer sections. Sections were deparaffined and boiled in the presence of 3% (w/v) sodium citrate with 0.2% (v/v) Triton X-100 to unmask antigen sites. Endogenous peroxidase activity was quenched with 1% (v/v) H2O2. Non-specific binding was avoided using a bovine serum albumin solution 1% (w/v). Sections were incubated with anti-GFAP at 1:5000 in PBS overnight at room temperature and then incubated with an anti-rabbit secondary antibody conjugated to HRP (DAKO kit, Dakocytomation). Finally, all sections were incubated with diaminobenzidine and co-stained with hematoxylin. Three sections were obtained from each brain and 5 different fields were analyzed in each section. Sections were analyzed field-by-field and determined with an image analyzer IM100 (Leica Cambridge, UK) [32] (link). All images correspond to dorsal striatum.
Dako kit
Manufactured by Agilent Technologies
Sourced in Denmark
The DAKO kit is a laboratory equipment product offered by Agilent Technologies. It is designed for use in various applications within the scientific and medical research fields. The core function of the DAKO kit is to provide a reliable and efficient solution for the analysis and detection of specific targets or analytes. The kit includes all the necessary components and reagents to facilitate the desired analysis or detection process. However, a more detailed and unbiased description of the kit's specific features and capabilities cannot be provided without the risk of extrapolation or interpretation.
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Lab products found in correlation
Ab19898, by Abcam (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Histofine, by Nichirei Biosciences (1 mentions)
Citrate buffer, by Agilent Technologies (1 mentions)
Dako envision, by Agilent Technologies (1 mentions)
Anti ar antibody, by Santa Cruz Biotechnology (1 mentions)
Sc 10, by Santa Cruz Biotechnology (1 mentions)
Phase contrast microscope, by Olympus (1 mentions)
Goat anti rabbit igg, by Agilent Technologies (1 mentions)
Diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Brdu in situ detection kit, by BD (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Scanscope at2, by Leica Biosystems (1 mentions)
Cytoseal mounting media, by Thermo Fisher Scientific (1 mentions)
Cy3 labeled anti rabbit igg antibody, by Jackson ImmunoResearch (1 mentions)
Fitc labeled anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions)
Variant machine, by Bio-Rad (1 mentions)
912 auto analyzer, by Hitachi (1 mentions)
15 protocols using dako kit
1
Immunohistochemical Quantification of GFAP
2
Immunohistochemical Analysis of Lung Tissue
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The left lung was removed and frozen in Tissue-Tek O.T.C. compound (Sakuar Finetek, Zoeterwoude, Netherlands). Frozen tissue blocks were cut to provide 4–6 μm sections, which were fixed in acetone for 5 min at −20°C and air dried. Endogenous peroxidase activity was blocked with immersion of sections in 0.3% H2O2, 0.1% NaN3 in methanol for 20 min at room temperature (RT) and washed three times. Nonspecific reactions were blocked with 10% FCS for 10 min at RT. After washes with PBS, sections were incubated with rat anti-Thy1 (catalog number 550543, BD Biosciences/Pharmingen, San Jose, CA, USA) for 2 h at RT. After PBS washing, sections were reincubated with universal immunoperoxidase polymer for mouse tissue sections in anti-rat primary antibody (Histofine, Nichirei Biosciences, Tokyo, Japan) for 30 min at RT and again reincubated for a 10 min in peroxidase substrate (Dako Kit, Dako, Glostrup, Denmark). The sections were counterstained with hematoxylin (Zymed Kit, Zymed Laboratories, San Francisco, CA) and mounting was added to the slides (Zymed Kit).
3
Immunohistochemical Profiling of Prostate Tissues
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IHC staining was performed on sections of paraffin-embedded tissues. All formalin-fixed prostate tissues were embedded in paraffin, and 5-µm sections were prepared. Sections were processed by heating at 70℃ in an oven for 30 min, dewaxing with xylene and alcohol (2 cycles, 10 min each), deparaffinization, and rehydration. Then endogenous peroxidase was blocked for 20 min in 0.3% hydrogen peroxide in water. The slides were then treated for antigen retrieval in a citrate buffer (pH 6) for 10 min at 95°C (DAKO PT Link, Glostrup, Denmark). The primary antibodies were anti-ESM1 (Abnova, H00011082-M02) and anti-AR antibody (Santa Cruz, sc-815). Samples were incubated with DAKO envision that contained horseradish peroxidase conjugated goat anti-rabbit, goat anti-mouse, or rabbit anti-goat antibodies (DAKO Cytomation, Glostrup, Denmark) for 30 min. The slides were visualized using a diaminobenzidine solution (DAB+; DAKO kit).
4
FGFR1 Amplification Assessment
5
Immunohistochemical Analysis of CEACAM5, EpCAM, and c-Met
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Based on hematoxylin–eosin-stained slides, a representative FFPE tissue block containing tumor and normal tissue from each patient was chosen by a board-certified pathologist (A.F.S.). After sectioning the FFPE blocks in 4 µm slides, these were mounted on adhesive slides (Starfrost), deparaffinized using xylene and rehydrated in decreasing concentrations of ethanol. Subsequently, slides were rinsed with distilled (DI) water and endogenous peroxidase was blocked with 0.3% hydrogen peroxidase (Merck Millipore) for 20 minutes. Slides were rinsed with DI water and antigen retrieval was performed in the DAKO PT LINK, Target Retrieval Solution pH 6.0 at 95°C for 10 minutes. After rinsing with phosphate buffered saline, slides were stained with predetermined dilutions using monoclonal antibodies (mAb) against CEACAM5 (clone CI-P83-1, SC-23928 from Santa Cruz Biotechnology, 0.2 µg/mL, dilution 1:2,500), EpCAM (clone MOC-31, Acris Antibodies, dilution 1:10,000) and a polyclonal antibody against c-Met (polyclonal rabbit, Santa Cruz SC-10, 1 µg/mL, dilution 1:100). After overnight incubation with the primary antibodies, slides were incubated with the secondary antibody (EnVision antimouse horseradish peroxidase [DAKO]) for 30 minutes, followed by diaminobenzidine solution (DAB+; DAKO Kit). All slides were counterstained with hematoxylin, dehydrated and finally mounted with pertex.
6
Histological and Immunohistochemical Analysis
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Briefly, fixed tissues and organs were embedded in paraffin. After being cut into 5 mm slices, the sections were stained using a standard H&E procedure. The results were analysed under a phase-contrast Olympus microscope (Olympus America, Inc.). For the immunohistochemical analysis, deparaffinised tumor sections underwent antigen retrieval in sodium citrate buffer (pH 6.0) using the high-pressure method. Then, the slides were blocked with 5% BSA and incubated with specific primary antibodies, followed by incubation with streptavidin–peroxidase horseradish peroxidase conjugated secondary antibodies and stained with a Dako kit (Dako, USA). The sections were counterstained with haematoxylin and analyzed under a phase-contrast Olympus microscope.
7
Immunostaining of Tumor Samples
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The mice were sacrificed and tumors were extracted at day 10. Immunohistochemical detection was performed using the DAKO kit (K5007, Denmark) following the manufacturer’s instructions. The primary antibody LC3 was 1:300 dilution (14600-1-AP, Proteintech), primary antibody p53 was 1:200 dilution (21891-1-AP, Proteintech), and primary antibody pAMPK was 1:100 dilution (CY6027, Abways). The secondary antibody was Goat-anti-Rabbit IgG (DAKO). After immunostaining, sections were counterstained with hematoxylin.
8
Immunohistochemical Analysis of Cell Markers
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Cut sections of 5 µm were prepared from formalin-fixed paraffin-embedded tissue for H&E staining or frozen tissue for immunohistochemistry staining. Frozen sections were fixed with acetone at −20 °C for 10 min and dried for 1 h at room temperature. BrdU staining was performed by using a BrdU in situ detection kit (BD Biosciences, San Jose, CA). The streptavidin–biotin–peroxidase method with the DAKO Kit (DAKO, Carpinteria, CA) was used to detect Ki67 and CD133 antigens. After inactivation of endogenous peroxidase and blocking of nonspecific antibody binding, the specimens were treated with anti-Ki67 (1:500, ab16667, Cambridge, MA) or biotinylated antibodies specific for CD133 (1:100, ab19898; Abcam) at 4 °C overnight. For Ki-67 staining, the tissue sections were subsequently incubated with biotin-conjugated goat anti rabbit IgG (1:200, #14708 S Cell Signalling, Danvers, MA) for 30 minutes at room temperature. Diaminobenzidine tetrahydrochloride (5 min, D4293, Sigma-Aldrich, St. Louis, MO) was used as the chromogen, and Mayer’s Hematoxylin (30 s, Dako, S3309) was used for counterstaining.
9
Immunohistochemical Analysis of Oral Cancer Stroma
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FFPE tumor blocks (n = 10) containing both the tumor stroma and tumor epithelia from oral cancer patients with lesion of buccal mucosa were used for immunohistochemical staining of COL1A2 and TIMP-1. Briefly, slides were deparaffinized for 30 min at 70 °C and passedthrough xylene, graded alcohol, and deionized water. Slides were then rinsed in low pH antigen retreival buffer, and microwaved at 60% power. After cooling, the slides were incubated in 3% hydrogen peroxide (#1,072,090,500; Supelco) for 10 min followed by rinsing with TBST and blocking for 30 min using 3% BSA and incubated with respective antibodies at room temperature for 60 min followed by rinsing with TBST and incubation with DAKO kit (#K5007; Dako; Agilent Technologies) for 30 min. Signal was detected using DAB followed by counterstain with hematoxylin solution. Slides were washed with xylene before being mounted using DPX mountant (#DAL1025; Qualigen Fine Chemicals TM; ThermoFisher Scientific) and observed under Nikon bright field microscope.
10
Immunohistochemistry of CD34 and CD133
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