ERG expression was evaluated using a commercial rabbit anti-ERG monoclonal antibody (cloneEP111; Dako, Denmark A/S). Deparaffinization, hydration of the slides, and blocking with pre-antibody solution (20 min) where performed in Dako PT Link (Code PT100/PT101). Then, a protocol template was created. The staining steps and incubation times were pre-programmed into the Autostainer Link software (Dako Autostainer). These were diluting anti-ERG primary antibody (1:50 for 20 min at room temp); applying Poly-HRP anti-rabbit IgG (20 min); applying DAB (20 min, Sigma Fast DAB tablets, Sigma-Aldrich, St. Louis MO); counter staining with EnVision FLEX hematoxylin (5 min); and dehydration, clearing, mounting, and covering.
Sigma fast dab tablets
Manufactured by Merck Group
Sourced in United States, Macao, Germany
Sigma Fast DAB tablets are a substrate for the detection of horseradish peroxidase (HRP) in immunohistochemistry and western blotting applications. The tablets contain 3,3'-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide, which react in the presence of HRP to produce a brown-colored precipitate.
Automatically generated - may contain errors
Lab products found in correlation
Autostainer link, by Agilent Technologies (1 mentions)
Cloneep111, by Agilent Technologies (1 mentions)
Ab26055, by Abcam (1 mentions)
Abc peroxidase kit, by Vector Laboratories (1 mentions)
Gad65, by Merck Group (1 mentions)
Sc 2027, by Abcam (1 mentions)
Vectastain elite kit, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
Antibody diluent, by Zytomed Systems (1 mentions)
Citric acid based antigen unmasking solution, by Vector Laboratories (1 mentions)
Vectastain elite abc hrp kit, by Merck Group (1 mentions)
Dpx mountant, by Merck Group (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Envision hrp kit, by Agilent Technologies (1 mentions)
Mab377, by Merck Group (1 mentions)
Imager z1 apotome microscope, by Zeiss (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Vectastain elite, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
10 protocols using sigma fast dab tablets
1
ERG Expression Immunohistochemistry Protocol
2
Immunohistochemistry of GABAA and GABAB Receptors
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Three-µm tissue sections were incubated with primary antibodies including 1∶50 to 1∶100 dilution of GABAA α1, β2/3 and π subunits, GABAB receptor R1 subtype (Cell-Signaling), R2 subtype (Cell Signaling), GAD67 (Santa Cruz, GAD65 (MILLIPORE) GAT2 (MILLIPORE) or ABAT. Immuno Shot reagents (COSMO BIO) were used to enhance the immune reaction in some assays. Chromogenic immunostaining was performed with an avidin–biotin immunoperoxidase kit (Vectastain Elite kit or ABC/Peroxidase kit, Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine (Sigma Fast DAB Tablets, Sigma-Aldrich). The kidneys were lightly counterstained with hematoxylin. Fluorescent immunostaining was performed using secondary antibody of Alexa Flour 568 conjugated goat anti-rabbit antibody and Alexa Flour 488 conjugated goat anti-mouse antibody (Life Technologies). GABAA π subunit antibody (Abcam: ab26055) was used for immunofluorescence studies. Normal rabbit IgG (Santa Cruz: sc-2027) and mouse monoclonal IgG1 (Abcam: ab81032) were used as negative control. Nuclei were stained by DAPI (Life Technologies). FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) was used for examination and image acquisition.
3
Immunohistochemical Staining of Paraffin-Embedded Tissue
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Formalin-fixed paraffin-embedded sections (4 μm) of omentum samples or co-cultures gels were heated for 1 hr at 60°C and then submerged twice in xylene for 5 min. Immunohistochemistry was performed using a Vectastain ABC Kit (Elite), as per the manufacturer's instructions. Slides were rehydrated and heated-antigen retrieval was performed using citric acid-based antigen unmasking solution (Vector Laboratories) and heated in a 2100 antigen-retriever (Aptum Biologics). Endogenous peroxidase activity was blocked using 3% H2O2 in PBS and sections were then blocked with 5% BSA. The primary antibody was added in antibody diluent (Zytomed) and covered at room temperature for 1 hr. Slides were incubated with a biotinylated secondary antibody (Vector) for 30 minutes. Subsequent steps were carried out according to the protocol included with the Vectastain Elite ABC HRP kit, after which the slides were incubated with DAB solution made using Sigmafast DAB tablets (Sigma-Aldrich). Finally, slides were counterstained in 50% Gill’s hematoxylin I, washed with water and dehydrated. After leaving to dry, coverslips were affixed using a drop of DPX mountant (Sigma-Aldrich).
4
Immunohistochemical Analysis of CEA in Colon Tissues
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Immunostaining of the colon tissues with CEA was performed as follows: the slides were deparaffinized, hydrated, blocked and then stained by CEA monoclonal antibody solution (sc-55547, Santa Cruz Biotechnology, Inc, USA) 1 : 50 dilution for 20 min. The slides were washed followed by applying the secondary antibody; HRP Envision kit, DAKO, Agilent, US (no dilution) for 20 min and applying 3,3′-diaminobenzidine (DAB, Sigma Fast DAB tablets, Sigma-Aldrich, St. Louis MO) for 20 min as previously mentioned.31 (link) The slides were finally counterstained with EnVision FLEX hematoxylin for 5 min, dehydrated, cleared and covered by a slip. The colonic sections were examined using a Zeiss light microscope (Oberkochen, Germany). The brownish-color was considered as a positive expression of CEA in the cells. Immunostaining expression was classified according to its distribution into either cytoplasmic type, in which immunostaining was homogeneously distributed in the cytoplasm of neoplastic cells or an apical type in which, there was predominant immunostaining on the cytoplasmic edge closest to the lumen of the neoplastic tissue cells.32 The positively stained cells were recorded within ten successive fields in each sample and quantified using Leica application suite for image analysis at 400× magnification.
5
Immunohistochemical Staining of Tumor Sections
6
Transthyretin Fibril Formation Assay
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Recombinant S52P TTR, 100 μl volumes at 0.5 mg/ml in 20 mm Tris-HCl containing 150 mm NaCl, 5 mm CaCl2, pH 7.4, containing 10 μm ThT (25 (link)) was incubated at 37 °C in Costar 96-well black plates in the presence of a protease at an enzyme:substrate ratio of 1:50. Plasmin, trypsin, thrombin, proteinase K, chymotrypsin, tryptase alpha, and kallikrein 12 were tested. The plate was sealed with clear sealing film and subjected to 900 rpm double-orbital shaking. Bottom fluorescence was recorded at 500-s intervals (BMG LABTECH, FLUOstar Omega). Homogenous 15% SDS-PAGE (GE Healthcare) under reducing conditions was used to analyze protein composition before and after fibril formation. After electrophoretic separation, samples treated and untreated with trypsin or plasmin were blotted onto an activated PVDF membrane. Western blotting was developed with polyclonal sheep anti-human TTR (6 μg/ml, The Binding Site, United Kingdom/code AU066X) and polyclonal rabbit anti-sheep peroxidase conjugate (1.3 μg/ml, Dako, Denmark/code P0163) as primary and secondary antibodies, respectively. Peroxidase activity was visualized using a precipitating substrate containing 3,3′-diaminobenzidine and urea hydrogen peroxide (SigmaFAST DAB tablets, Sigma-Aldrich).
7
Neuronal Nuclei Immunohistochemistry in Brain Tissue
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All brain tissues used for imaging was sectioned as previously described (Morganti 2014). 40 μm free-floating sections were stained for neuronal nuclei (NeuN, MAB377, Millipore) with biotinylated secondary antibodies (Biotinylated anti-mouse IgG, BA-2001, Vector) and revealed with DAB (Sigmafast DAB tablets, Sigma-Aldrich). Sections were mounted onto Superfrost Plus slides (Fisher #12-550-15). All imaging was achieved using a Zeiss Imager.Z1 Apotome microscope controlled by ZEN software (Zeiss 2012).
8
Immunohistochemical Staining Protocols
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The immunohistochemical methods have been described in detail previously (26 (link), 27 (link), 30 (link)). Briefly, tissues were cryo-sectioned at a thickness of 20 microns and were collected serially in groups of 5, and placed in 24-well tissue culture plates in the same solution. Endogenous peroxidase was blocked with a 50:50 mixture of methanol and water containing 1% H2O2 for 30 min with agitation. Tissue sections were incubated overnight with the Quin antibodies, diluted 1:10,000, using constant rotary agitation. The antibodies were visualized by the avidin–biotin complex method with HRP as the enzyme marker (Vectastain Elite, Vector Labs). The sections were incubated with the biotinylated secondary antibody and avidin–biotin–peroxidase complex solutions for 90 min each, and then developed with diaminobenzidine and urea peroxide as chromogen and substrate (Sigmafast DAB tablets, Sigma) for 10–15 min. Lectin histochemistry with Griffonia simplicifolia-B4 lectin (GSL-IB4) and double staining with Quin was done as described earlier (27 (link)).
9
Immunohistochemical Brain Tissue Analysis
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Stainings were performed as described24 . Mice were anesthetized (Ketamine/Xylazine/Heparine) and transcardially perfused with 4% paraformaldehyde (PFA). Brains were dissected and post-fixed in 4% PFA for a minimum of 24 h, transferred to 30% sucrose for cryoprotection for at least 48 h, and embedded in 5% low melting point Agarose (Roth, Germany). 30 µm sagittal brain sections were prepared (VT1200s Leica Microsystems) and, after peroxidase block (1% H2O2 in PBS, 30 min RT), blocked with 10% Normal Goat Serum (Dako, Germany) and 0.1% Triton X-100 (Sigma, Germany)in PBS (1 h, RT), and incubated with primary antibody overnight at 4 °C. After incubation with biotinylated secondary antibodies (1.5 h at RT), proteins were visualized using the ABC method with the Vectastain ABC kit (Vector Laboratories) and the DAB substrate Sigma Fast DAB Tablets (Sigma-Aldrich, Germany). Brain slices were mounted on glass slides and counter-stained with haematoxylin (Roth), followed by dehydration (70%, 80% and 100% ethanol, xylene) and embedding with phenol-free Kaiser’s glycerol gelatine (Roth). Images were taken with a Zeiss AxioCam MR coupled to a Zeiss Axio Imager M2 using the Zeiss Axiovision software.
10
Immunohistochemical Analysis of TGF-β1
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Immunohistochemistry was carried out on paraffin-embedded sections using mouse monoclonal anti-TGF-β1 (3C11, Santa Cruz Biotechnology; 1:50 dilution); an adequate anti-mouse secondary antibody was then used following manufacturer’s recommendations. Signal was revealed with standard immunohistochemistry methods and SIGMAFAST™ DAB Tablets (Sigma-Aldrich, D0426, Merck Life Science S.L.U.) as a chromogen. Slides were co-stained with haematoxylin and photographed with an Olympus BX41 microscope (Olympus Iberia S.A.U.).
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