All tissue samples were obtained with full ethical approval from the KIRAMS institutional review board (IRB No. K-1103-003-016). Immunohistochemical staining for SOCS1, SOCS3, and SOCS5 were performed by an automated immunohistochemical stainer (Autostainer 480, Thermo Fisher Scientific, Fremont, CA, USA) on formalin-fixed paraffin embedded tissue sections of squamous cell carcinomas obtained from uterine cervix. Sections were deparaffinized with xylene for 15 min and pretreated in a microwave oven using 0.01 M citrate buffer (pH 6.0) for 30 min. Sections were incubated with primary antibodies directed against SOCS1 (1:25, ab62584, Abcam, Cambridge, UK), SOCS3 (1:100, ab16030, Abcam) and SOCS5 (1:100, sc-100858, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at room temperature and detected using UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific).
Ultravision lp detection system hrp polymer dab plus chromogen
Manufactured by Thermo Fisher Scientific
Sourced in United States
The UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen is a laboratory equipment product designed for immunohistochemistry (IHC) applications. It is a polymer-based detection system that uses horseradish peroxidase (HRP) and 3,3'-Diaminobenzidine (DAB) as the chromogen for visualization of target antigens in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Autostainer 480, by Thermo Fisher Scientific (2 mentions)
Sc 100858, by Santa Cruz Biotechnology (1 mentions)
Ab16030, by Abcam (1 mentions)
Ab62584, by Abcam (1 mentions)
Dmlb microscope, by Leica (1 mentions)
Dc200, by Leica (1 mentions)
Paraffin, by Bio-Optica (1 mentions)
Ab53519, by Abcam (1 mentions)
Antigen retrieval pt module, by Thermo Fisher Scientific (1 mentions)
Entellan, by Merck Group (1 mentions)
Icc50 hd camera, by Leica (1 mentions)
Dm2000 led, by Leica (1 mentions)
Specific staining kits, by Bio-Optica (1 mentions)
5 protocols using ultravision lp detection system hrp polymer dab plus chromogen
1
Immunohistochemical Analysis of SOCS Proteins in Cervical SCC
2
Immunohistochemical Analysis of Oxidative Stress
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Aortic tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin (Bio-Optica Milano SpA, Milan, Italy), then sliced into serial 4 m-thick sections and placed on poly-L-lysine coated glass slides (Menzel-Glaser, Brunswick, Germany). Slides were deparaffinised, rehydrated and immersed in 10 mM citric acid, pH 6, in a microwave oven (VWR International PBI Srl, Milan, Italy) to exclude epitope masking owing to fixation. Sections were immunostained with primary antibodies against SOD3 (1:10), Akt1 (1:100) and phospho-Akt1/2/3 (1:50) and detected with an indirect immunoperoxidase technique employing UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s protocol. As negative controls, sections were incubated with PBS solution (Bio-Optica Milano SpA). Nuclear staining was carried out using hematoxylin. Microscopic analysis was performed with a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany). Pictures were taken in bright field with a digital camera (Leica DC200; Leica Microsystems) connected to the microscope.
3
Immunohistochemical Analysis of Vimentin, Snail, and TGF-β2
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For immunohistochemistry (IHC), 4-µm sections were dewaxed, hydrated and heat-treated in 1 mM EDTA pH 8 in an antigen retrieval PT module (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 95 °C for 20 min. Sections were then stained with the following antibodies: Rabbit monoclonal antibody to vimentin (Clone SP20) 1:200 dilution (Spring Bioscience Corp., Pleasanton, CA, USA); goat polyclonal antibody to Snail (ab53519), 1:200 dilution (Abcam, Cambridge, MA, USA); mouse monoclonal antibody against TGF-β2 (220ct16.4.3.1), 1:40 dilution (Cell Marque Corp., Rocklin, CA, USA). The staining procedure was performed according to the manufacturers’ protocols using an Autostainer 480 (Thermo Fisher Scientific Ltd., Vantaa, Finland). The visualization system included UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific Inc., Waltham, MA, USA) and hematoxylin counterstaining. For the negative control sections, primary antibodies were replaced with phosphate-buffered saline and processed in the same manner. Stained slides were evaluated by two pathologists (MS and OT). The number of Snail and TGF-β2 positive cells was counted in 10 randomly selected fields by light microscopy at 400× magnification. The percentage of positive cells was expressed relative to the total number of tumor or epithelial cells counted.
4
Melanoma Tissue Array Immunohistochemistry
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The tissue array used in this study, referred to as MEL961, includes 48 tissue sections of melanomas at different stages and also normal tissue (http://www.pantomics.com/TissueArrays/Skin/Human/MEL961.aspx ). Tissue arrays were deparaffinated and hydrated following standard procedures. Antigenicity was recovered treating samples with Retrieval solution (S1699; DACO, Rune Linding, Denmark) at 100°C for 20 min. After washing, tissue arrays were blocked with goat serum 2% (diluted in PBS 1×) for 30 min and incubated with the primary antibody (1 : 100 in goat serum 2%) overnight. Samples were then washed 3× for 5 min with PBS 1×. First, antibodies were subsequently detected using the UltraVision LP Detection System-HRP Polymer & DAB Plus Chromogen (TL-015-HD; Thermo Scientific, West Palm Beach, Florida, USA) following the instructions provided by the manufacturer. Finally, samples were counterstained with haematoxylin, dehydrated and mounted in permanent medium Entellan (Merck, Darmstadt, Germany).
5
Histological Analysis of Decellularized Human Skin
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Specimens of native skin (n = 8) and d-HuSk (n = 8) were fixed in 10% neutral-buffered formalin, dehydrated in a graded series of alcohols, embedded in paraffin, then sliced into serial 5-μm-thick sections. Following standard protocols, after rehydration sections were stained with Hematoxylin and Eosin to evaluate the effectiveness of decellularization, and with Paraldehyde fuchsin Gomori or Alcian blue to detect elastic fibers or GAGs, respectively, by specific staining kits (both from Bio-Optica, Milan, Italy). For the immunodetection of collagen, fibronectin, tenascin, and laminin, sections of d-HuSk were deparaffinized, rehydrated, and immunostained using indirect immunoperoxidase technique. The presence and localization of antigen ⋅ antibody complexes were revealed by the UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Scientific, Waltham, MA, United States), as previously described (Di Meglio et al., 2017 (link)). Stained sections were evaluated and documented by at least three independent observers using a light microscope DM2000 Led (Leica Microsystems) equipped with an ICC50HD camera (Leica Microsystems).
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