Post imaging, the samples were fixed in 10% formalin and embedded in paraffin, sectioned into 4-μm-thick slices, and mounted on microscope slides, according to standard practice. All processing and staining procedures were performed by the Tufts Medical Center pathology services using an automated staining machine (Benchmark Ultra, Ventana Medical Systems, Inc., Tucson, AZ) according to established protocols. Typical hematoxylin and eosin (H&E) along with immunohistochemical (IHC) staining for Ki67 and p16 markers was performed. For IHC, anti-Ki-67 (30–9) Rabbit Monoclonal Primary and CINtec® p16 Histology Ventana-ready-to-use antibodies were utilized. The IHC sections were counterstained with hematoxylin and a bluing agent by using 3–3′-diaminobenzidine detection kit (Ventana Medical Systems, Inc.). Control slides of human tonsil and cervical carcinoma were used to confirm the sensitivity and specificity of the Ki-67 and p16 staining respectively. Digital images were acquired with a BZ-X710 Keyence microscope equipped with a 40x (NA 0.6) Nikon objective. Acquisition settings were held constant for all captured images per antibody group. Visual interpretation of the staining intensities in conjunction with histological examination was performed by the study’s board-certified pathologist (E.G).
3 3 diaminobenzidine detection kit
Manufactured by Roche
The 3–3′-diaminobenzidine detection kit is a laboratory reagent used for the detection and visualization of specific proteins or enzymes in biological samples. This kit provides the necessary components for the enzymatic conversion of 3–3′-diaminobenzidine into a colored precipitate, which can be observed under a microscope.
Automatically generated - may contain errors
3 protocols using 3 3 diaminobenzidine detection kit
1
Immunohistochemical Analysis of Ki-67 and p16
2
Immunohistochemical Evaluation of Tumor Samples
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Post imaging, the samples were fixed in 10% formalin and embedded in paraffin, sectioned into 4-μm-thick slices, and mounted on microscope slides, according to standard practice. All processing and staining procedures were performed by the Tufts Medical Center pathology services using an automated staining machine (Benchmark Ultra, Ventana Medical Systems, Inc., Tucson, AZ) according to established protocols. Typical hematoxylin and eosin (H&E) along with immunohistochemical (IHC) staining for Ki67 and p16 markers was performed. For IHC, anti-Ki-67 (30-9) Rabbit Monoclonal Primary and CINtec® p16 Histology Ventana-ready-to-use antibodies were utilized. The IHC sections were counterstained with hematoxylin and a bluing agent by using 3-3′-diaminobenzidine detection kit (Ventana Medical Systems, Inc.). Control slides of human tonsil and cervical carcinoma were used to confirm the sensitivity and specificity of the Ki-67 and p16 staining respectively. Digital images were acquired with a BZ-X710 Keyence microscope equipped with a 40x (NA 0.6) Nikon objective. Acquisition settings were held constant for all captured images per antibody group. Visual interpretation of the staining intensities in conjunction with histological examination was performed by the study’s board-certified pathologist (E.G).
3
Immunohistochemical Detection of Id1 and CD31
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Immunohistochemical detection was done with a Discovery XT system (Ventana Medical Systems,Tucson, AZ). Slides were blocked with 10% normal goat serum and 2% bovine serum albumin for 30 minutes. Primary antibody incubation was done for 2 hours with anti- Id1 rabbit monoclonal 195–14 (diluted 1:500) followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA; 1:200 dilution) for 8 minutes. Endogenous biotin blocking kit, blocker D, streptavidin-horseradish peroxidase, and 3,3-diaminobenzidine detection kit were used according to the manufacturer’s instructions (Ventana Medical Systems). Immunostaining for CD31 was performed on a Leica Bondtrademark RX using the Bondtrademark Polymer Refine Detection Kit (Cat. No. DS9800). The sections were pre-treated using heat mediated antigen retrieval with EDTA (pH9, Epitope Retrieval Solution 2, Cat. No. AR9640) for 20mins. The sections were then incubated with anti-Mouse CD31 (PECAM-1) antibody (Dianova, Cat. No. DIA-310) diluted at 1:250 for 30mins. DAB was used as the chromogen. The sections were then counterstained with hematoxylin and mounted.
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