Each human PC tumor case was thoroughly reviewed, formalin-fixed, and paraffin-embedded blocks were acquired within Department of Surgery, University of Alabama at Birmingham, and Dept. of Pathology, NICHD followed by the protocol described previously using DAKO immunohistochemistry kit (Pozo et al., 2013 (link)). Human adrenal tumor tissue microarray (US Biomax Inc.) was de-waxed at 60°C for 2 h followed by standard IHC protocol. Primary antibodies used included those for Cdk5 (Rockland), -p35/25 (Cell Signaling), ChrA (Abcam), anti-tyrosine hydroxylase (Abcam), and GFP (Cell Signaling). Secondary antibody alone was used as negative control. Quantitative analysis of DAB stained images were performed by using optical density (color deconvolution algorithm) within IHC profiler plug-in compatible with ImageJ digital image analysis software (Varghese et al., 2014 (link)).
Immunohistochemistry kit
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The Immunohistochemistry kit by Agilent Technologies is a set of laboratory reagents and tools designed for the detection and visualization of specific proteins or antigens in tissue samples. The kit provides the necessary components for the immunohistochemical staining process, allowing researchers to identify the presence and distribution of target molecules within fixed and sectioned tissue specimens.
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5 protocols using immunohistochemistry kit
1
Immunohistochemical Profiling of Adrenal and Prostate Tumors
2
Immunohistochemical Analysis of Hedgehog Pathway
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Mouse anti-human SHH (BD-610182) primary antibody was purchased from American BD company, mouse anti-human SMO (SC-8424), PCNA (SC-6260), GLI1 (SC-8598), PTC-1 (SC-3039), PTC-2 (SC-4853) primary antibody was purchased from Santa Cruz, USA, and immunohistochemistry kit was purchased from Dako, USA.
3
Immunohistochemical Evaluation of pErk in ESCC
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ESCC tissue microarray containing 15 paired cancer and para-tumour tissues was purchased from the OUTDO Bio Tech Co. (Shanghai, China). Sections were dewaxed by xylene before heated for 10 min at 95 °C in a microwave oven for antigen retrieval. Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2 solution for 10 min. The sections were then incubated with antibodies against pErk (1:1000) at 4 °C overnight. Immunohistochemical staining was performed using a immunohistochemistry kit (Dako, Copenhagen, Denmark). No significant staining was observed in the negative controls, which were prepared by using the same class of immunoglobulin at the same concentration. To evaluate pErk protein expression, both the extent and intensity of immunoreactivity were assessed and scored, in which the scores of the extent of immunoreactivity ranged from 0 to 3 according to the percentage of cells that had positive staining in each microscopic field of view (0, <25%1, 25–50% 2, 50–75% 3, 75–100%) while the scores of intensity were as follows: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. A total score was obtained by multiplying the scores for extent and intensity.
4
Immunohistochemical Analysis of Tight Junctions
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Liver and colon tissue sections were stained using an immunohistochemistry kit (DAKO, Denmark) as previously described[20 (link)]. The NanoZoomer digital pathology scanner was used to scan the stained sections into a format suitable for analysis. Each section was randomly selected at 200× and 400× magnifications for analysis. Image-Pro Plus software was used to measure the average optical density of tight junction protein-1 (ZO-1).
5
Immunohistochemical Analysis of Tumor Biomarkers
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We performed IHC to evaluate the clinical and animal tumor samples. Paraffin sections with a thickness of 5 µm were deparaffinized with xylene and absolute ethanol. Antigen recovery was performed after washing with distilled water. The paraffin sections were incubated with 3% hydrogen peroxide at room temperature (22 °C) for half an hour and blocked with bovine serum albumin for the same duration followed by overnight probing with the primary antibody. anti-HER2 (catalog number AF7681, 1:100 dilution, Affinity Biosciences), rabbit anti-p-HER2/ERBB2 (Tyr1248, catalog number AF3069, 1:100 dilution, Affinity Biosciences). anti-p-TSC2 (Thr1462, catalog number AF3334, 1:50 dilution, Affinity Biosciences). anti-p-mTOR (Ser2448, catalog number 5536, 1:100 dilution, Cell Signaling Technology), rabbit anti-p-P70S6K (Thr389, catalog number AF3228, 1:100 dilution, Affinity Biosciences). Finally, the sections were washed thrice with PBS. The sample was then incubated with horseradish peroxidase-linked secondary antibody from the immunohistochemistry kit (Dako, catalog number K5007, Copenhagen, Denmark). After incubating for 1 h and washing thrice, diaminobenzidine solution was added. The sections were then stained and counterstained with hematoxylin, dehydrated, and observed under a fluorescence microscope.
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