Control and null embryos were harvested at E11.5, fixed, dehydrated, embedded in paraffin, sectioned at 5μm as described for laminin staining and subject to immunohistochemistry for E-cadherin. Antigen retrieval was performed using a heat induced citrate buffer, slides were rinsed in 3% hydrogen peroxide/PBS to block endogenous peroxidase, washed in 0.5% Triton X-100/PBS, nonspecific background staining blocked using 10%BSA incubation of 60 minutes, and incubated overnight at 4°C at 1:50 concentration using monoclonal mouse anti-E-cadherin antibody (BD Pharmingen catalog #610181). Slides were then incubated in Mach 2 goat anti-mouse IgG HRP conjugated polymer (Biocare Medical catalog #MHRP520H) for 40 minutes, developed with DAB (Dako’s DAB substrate-chromogen system catalog #K3466) for two minutes, washed, and counterstained with hematoxylin (Sigma-Aldrich catalog #HHS128). Slides were imaged on a Nikon® Eclipse 80i light microscope.
Dab substrate chromogen system
Manufactured by Merck Group
Sourced in Japan
The DAB substrate-chromogen system is a laboratory reagent used for the visualization and detection of targeted proteins or antigens in various immunohistochemical and immunocytochemical applications. The system utilizes 3,3'-diaminobenzidine (DAB) as the chromogenic substrate, which undergoes an enzymatic reaction to produce a brown-colored precipitate at the site of the target analyte. This product enables the specific labeling and detection of relevant biomolecules in research and diagnostic settings.
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4 protocols using dab substrate chromogen system
1
E-cadherin Immunohistochemistry in Mouse Embryos
2
Amylase and CK-19 Double Staining Protocol
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A sequential method for amylase/CK-19 double staining was used according to the immunohistochemistry enzyme double staining protocol described in a previous study (34 (link)). Briefly, the sections were incubated with goat polyclonal anti-CK19 antibody (dilution 1:100; Santa Cruz Biotechnology) as the first primary antibody and detected using the DAB substrate chromogen system (Sigma). The sections were then blocked again with normal serum, and incubated with the second primary antibody, mouse monoclonal anti-amylase antibody (dilution 1:100; Santa Cruz Biotechnology), after incubating with the anti-mouse secondary antibody and avidin-biotin-peroxidase complex; 3-amino-9-ethylcarbazole (AEC) peroxidase substrate with a characteristic red color was used to detect positive staining and to distinguish from the brown color of DAB. The negative control was established by replacement of the primary antibody with normal serum. Specific antibody-labeled signals were analyzed under a microscope (Nikon, Tokyo, Japan).
3
Immunohistochemical Analysis of FAPI Expression
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Three out of the 31 patients undergoing 68Ga-FAPI-PET/CT provided sufficient material for the conduction of immunohistochemistry. All slices were obtained by the Tissue Bank of the National Center for Tumor Diseases (NCT) Heidelberg, Germany in concordance with the regulations of the tissue bank and the approval of the ethics committee of Heidelberg University. Immunhistochemistry was performed on 0.5-μm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections. As primary anti-FAP antibody, we utilized ab207178 (EBR20021; Abcam, Cambridge, UK), diluted 1:200. The slices were pretreated with the Buffer Substrate DAKO (pH 9, 10x, S2375) at 95° for 20 min, followed by incubation with the primary antibody at 4°C overnight. After that, we stained with the secondary antibody (DAKO EnVision and System-HRP labeled polymer anti-rabbit, code K4003) for 45 min at room temperature. In terms of visualization, Universal DAB Detection Kit (Dako Liquid DAB + Substrate Chromogen System, K3468) was utilized and counterstaining was conducted with Mayer’s hemalaun (Mayer’s Haemalaunsolution, Merck KGaA) for 10 s. Afterwards images were scanned and subsequently digitalized with the help of NanoZoomer S60 Digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan).
4
Multimodal Protein Expression Analysis in Teratocarcinoma
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Cell lysates were separated by 12% SDS-PAGE and immunoblotted with the antibodies against Oct4 (1:500, Santa Cruz), VGLUT2 (1:500, Santa Cruz), GLS (1:500, Abcam), or EAAT1 (1:500, Abcam). The membranes were then incubated with corresponding secondary antibodies. The immunoblots were visualized and scanned using an imaging system (Odyssey FC, LI-COR Biosciences).
Immunohistochemical staining was performed on formalin-fixed preparations from human teratocarcinoma tissue slices obtained from the tissue bank of Shanghai Cancer Hospital. The tissue slices were incubated with primary antibodies and subsequently with secondary antibodies. After incubation with VECTASTAIN® ABC Reagent for 30 min, peroxidase activity was developed with DAB Substrate–Chromogen System (Merck). After a final wash in PBS, the slices were counterstained with hematoxylin. Immunofluorescence staining was performed on stem cells and mouse transplanted teratocarcinoma tissue slices. They were incubated with corresponding antibodies. In some cases, dual immunofluorescence staining was performed to investigate whether the transmission components colocalized in the same cells. The primary antibodies used were anti-Oct4 (1:500), anti–GLS (1:100), anti-VGLUT2 (1:100), and anti-EAAT1 (1:500).
Immunohistochemical staining was performed on formalin-fixed preparations from human teratocarcinoma tissue slices obtained from the tissue bank of Shanghai Cancer Hospital. The tissue slices were incubated with primary antibodies and subsequently with secondary antibodies. After incubation with VECTASTAIN® ABC Reagent for 30 min, peroxidase activity was developed with DAB Substrate–Chromogen System (Merck). After a final wash in PBS, the slices were counterstained with hematoxylin. Immunofluorescence staining was performed on stem cells and mouse transplanted teratocarcinoma tissue slices. They were incubated with corresponding antibodies. In some cases, dual immunofluorescence staining was performed to investigate whether the transmission components colocalized in the same cells. The primary antibodies used were anti-Oct4 (1:500), anti–GLS (1:100), anti-VGLUT2 (1:100), and anti-EAAT1 (1:500).
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