Stainings were performed on sections from brain tissues fixed in formalin and treated with concentrated formic acid to inactivate prions. Partially protease-resistant prion protein deposits, astrogliosis and microglia deposition were visualized by staining brain sections with the SAF84 antibody (1:200, SPI bio), GFAP (1:1000, Millipore) and IBA1 (1:2500, WAKO) respectively on a NexES immunohistochemistry robot (Ventana instruments) using an IVIEW DAB Detection Kit (Ventana), after preceding incubation with protease 1 (Ventana). Images of DAB stained sections were acquired using the NanoZoomer scanner (Hamamatsu Photonics) and NanoZoomer digital pathology software (NDPview; Hamamatsu Photonics). Quantifications of IBA1, GFAP staining and vacuoles in mouse sections were performed on acquired images; regions of interest were drawn on a Digital Image Hub (Leica Biosystems) and analyzed as previously described [77 ].
Protease 1
Manufactured by Roche
Sourced in Switzerland
Protease 1 is a laboratory equipment product manufactured by Roche. It is a versatile enzyme that breaks down proteins into smaller peptides or amino acids. The core function of Protease 1 is to facilitate protein analysis and sample preparation in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Iview dab detection kit, by Roche (4 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Nanozoomer scanner, by Hamamatsu Photonics (2 mentions)
Axiophot light microscope, by Zeiss (2 mentions)
Ez prep solution, by Roche (2 mentions)
Optiview dab detection kit, by Roche (2 mentions)
Digital image hub, by Leica (1 mentions)
Nanozoomer digital pathology software ndpview, by Hamamatsu Photonics (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Antibody diluent, by Abcam (1 mentions)
Ventana discovery ultra, by Roche (1 mentions)
Discovery wash solution, by Roche (1 mentions)
Biotin conjugated donkey anti goat secondary antibody, by Jackson ImmunoResearch (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Abc complex solution, by Vector Laboratories (1 mentions)
Anti iba1 antibody, by Fujifilm (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Ventana discovery xt, by Roche (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
F4 80, by Abcam (1 mentions)
14 protocols using protease 1
1
Immunohistochemical Analysis of Prion Protein
2
Automated Immunohistochemistry for Annexin V
3
Immunohistochemical Analysis of GPNMB in Colorectal Cancer
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Clinical samples for IHC were obtained from CRC patients who received treatment at Gifu University (Gifu, Japan) from January 2010 to January 2016. Seven sets of naive primary tumors and remaining liver metastatic tumors after chemotherapy regimens, such as folinic acid, fluorouracil, and oxaliplatin (FOLFOX) containing anti-EGFR therapy, were used for this analysis 2 (link),3 (link). Informed consent was obtained from each patient.
IHC for GPNMB was performed according to standard procedures using the same antibody described here and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA) 19 (link). Goat IgG, a polyclonal isotype control (Abcam), was used as a negative control. For preliminary experiments, we created cell blocks containing three cell lines (GPNMB expression levels: high, SK-BR-3; moderate, MKN1; and low, LS174T). We used PROTEASE 1 (Ventana, Tuscon, AZ, USA) for antigen retrieval and determined the best conditions for GPNMB IHC (Fig.S1 ). Changes in GPNMB-staining intensity between each primary tumor and metastasis were determined by an experienced pathologist. This study was approved by the Central Ethics Committee of Gifu University.
IHC for GPNMB was performed according to standard procedures using the same antibody described here and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA) 19 (link). Goat IgG, a polyclonal isotype control (Abcam), was used as a negative control. For preliminary experiments, we created cell blocks containing three cell lines (GPNMB expression levels: high, SK-BR-3; moderate, MKN1; and low, LS174T). We used PROTEASE 1 (Ventana, Tuscon, AZ, USA) for antigen retrieval and determined the best conditions for GPNMB IHC (Fig.
4
Prion Inactivation and Immunohistochemistry
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Formalin-fixed tissues were treated with concentrated formic acid for 60 min to inactivate prion infectivity and embedded in paraffin. Paraffin sections (2mm) of brains were stained with hematoxylin/eosin (HE). After deparaffinization through graded alcohols, antibodies GFAP (1:300; DAKO, Carpinteris, CA) for astrocytes were applied and visualized using standard methods, IBA-1 (1:1000; Wako Chemicals GmbH, Germany) was used for highlighting activated microglial cells. Stainings were visualized using DAB (Sigma-Aldrich), with a hematoxylin counterstain applied subsequently. For partially protease K-resistant prion protein deposition staining, deparaffinized sections were incubated for 6 min in 98% formic acid and washed in distilled water for 30 min. Sections were incubated in Ventana buffer and stains were performed on a NEXEX immunohistochemistry robot (Ventana instruments, Switzerland) using an IVIEW DAB Detection Kit (Ventana). After incubation with protease 1 (Ventana) for 16 min, sections were incubated with anti-PrP SAF-84 (SPI bio, A03208, 1:200) for 32 min. Sections were counterstained with hematoxylin. Sections were imaged using a Zeiss Axiophot light microscope.
5
Immunohistochemical Analysis of Prion Protein
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Formalin-fixed tissues were treated with concentrated formic acid for 60 minutes at room temperature to inactivate prion infectivity. Tissue was embedded in paraffin and cut into 2 μm sections. After deparaffinization through graded alcohols sections were stained with hematoxylin/eosin. Antibody SAF-84 (A03208, 1:200, SPI-Bio, Waterloo, Australia) was used to detect partially protease-resistant prion protein deposition on a NEXES immunohistochemistry robot (Ventana Instruments, Basel Switzerland) using an IVIEW DAB Detection Kit (Ventana), after incubation with protease 1 (Ventana). Microglia was detected using anti-Iba 1 antibody (WAKO). Sections were deparaffinized through graded alcohols and heat-induced antigen retrieval was performed in citrate buffer (0.01 M; pH 6). Sections were incubated with anti-Iba1 Ab (1∶2500). Stainings were visualized using DAB (Sigma-Aldrich) and H2O2 (Sigma-Aldrich), after incubation with a biotinylated secondary antibody (Vector Laboratories) followed by the ABC complex solution (Vector laboratories). Sections were counterstained with Hematoxylin. Images of HE and DAB stained sections were acquired using a NanoZoomer scanner (Hamamatsu Photonics) and NDPview digital pathology software (Hamamatsu Photonics).
6
Immunohistochemical Staining of Tissue Samples
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The histological specimens were embedded in paraffin, sectioned (4-μm slices) and stained with haematoxylin & eosin. For IHC, slides were stained using a Ventana Discovery XT automated system (Ventana Medical Systems). Briefly, slides were deparaffinised on the automated system with EZ Prep solution (Ventana). Enzymatic retrieval method was used in Protease 1 (Ventana). The rabbit primary antibodies that react to F4/80, α-smooth muscle actin (α-SMA) and CD206 (all purchased from Abcam) were used at 1:400, 1:250 and 1:1200 dilutions, respectively, in Dako antibody diluent (Agilent) and incubated for 60 min. The Ventana OmniMap Anti-Rabbit Secondary Antibody was used for 8 min. The detection system used was the Ventana ChromoMap Kit, and slides were then counterstained with haematoxylin, followed by dehydrated and cover-slipping.
7
Histological analysis of prion-infected brains
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Prion-infected brain tissues were harvested and fixed in formalin, followed by treatment with concentrated formic acid for 60 min to inactivate prion infectivity and embedded in paraffin.
Paraffin sections (2 μm) of brains were stained with hematoxylin/eosin (HE) to visualize prioninduced lesions and vacuolation. For the histological detection of partially proteinase Kresistant prion protein deposition, deparaffinized sections were pretreated with formaldehyde for 30 min and 98% formic acid for 6 min, and then washed in distilled water for 30 min.
Sections were incubated in Ventana buffer and stains were performed on a NEXEX immunohistochemistry robot (Ventana instruments, Switzerland) using an IVIEW DAB Detection Kit (Ventana). After incubation with protease 1 (Ventana) for 16 min, sections were incubated with anti-PrP SAF-84 (1:200, SPI bio, A03208) for 32 min. Sections were counterstained with hematoxylin. To detect astrogliosis and microglial activation, brain sections were deparaffinized through graded alcohols, anti-GFAP antibody (1:300; DAKO, Carpinteria, CA) were applied for astrogliosis, anti-Iba1 antibody (1:1000; Wako Chemicals GmbH, Germany) was used for highlighting activated microglial cells. Stainings were visualized using DAB (Sigma-Aldrich), hematoxylin counterstain was subsequently applied. Sections were imaged using a Zeiss Axiophot light microscope.
Paraffin sections (2 μm) of brains were stained with hematoxylin/eosin (HE) to visualize prioninduced lesions and vacuolation. For the histological detection of partially proteinase Kresistant prion protein deposition, deparaffinized sections were pretreated with formaldehyde for 30 min and 98% formic acid for 6 min, and then washed in distilled water for 30 min.
Sections were incubated in Ventana buffer and stains were performed on a NEXEX immunohistochemistry robot (Ventana instruments, Switzerland) using an IVIEW DAB Detection Kit (Ventana). After incubation with protease 1 (Ventana) for 16 min, sections were incubated with anti-PrP SAF-84 (1:200, SPI bio, A03208) for 32 min. Sections were counterstained with hematoxylin. To detect astrogliosis and microglial activation, brain sections were deparaffinized through graded alcohols, anti-GFAP antibody (1:300; DAKO, Carpinteria, CA) were applied for astrogliosis, anti-Iba1 antibody (1:1000; Wako Chemicals GmbH, Germany) was used for highlighting activated microglial cells. Stainings were visualized using DAB (Sigma-Aldrich), hematoxylin counterstain was subsequently applied. Sections were imaged using a Zeiss Axiophot light microscope.
8
Automated Immunohistochemistry for Androgen Receptor
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Immunohistochemical staining was performed by an automated slide staining instrument BenchMark ULTRA (Ventana Medical System/Roche, Tucson, Arizona, United States). For immunohistochemistry, slides were deparaffinized and rehydrated. AR antibody clone M AR 441 (Dako, Glostrup, Denmark) was used as primary antibody. For detection of the AR, tissues were pretreated by heat antigen retrieval with a Cell Conditioner 1 solution (Roche, Basel, Switzerland) for 64 min with protease 1 (Roche). AR antibodies were diluted 1:200 in an antibody-diluent and incubated for 32 min at 37 °C in the platform. For visualization, the indirect biotin-free OptiView DAB Detection Kit (Roche) was used. The slides were counterstained and mounted. Internal controls served as positive controls for AR.
In microscopic assessment, AR staining was distributed homogeneously. Expression was quantified according to a modified scoring system, which has already been used for assessment of AR immunoreaction. Diversity of positive cells was classified to a score 0–5 [23] (link) by a researcher blinded for diabetes status.
In microscopic assessment, AR staining was distributed homogeneously. Expression was quantified according to a modified scoring system, which has already been used for assessment of AR immunoreaction. Diversity of positive cells was classified to a score 0–5 [23] (link) by a researcher blinded for diabetes status.
9
Multiplex IHC Analysis of Immune Markers
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The IHC staining was performed on the Ventana Benchmark XT from Roche with the Ultra View Universal DAB Kit using a rabbit antibody against CD3 and mouse antibodies against CD8, pan-cytokeratin and Foxp3. Briefly, the slides were deparaffinized using deparaffinization solution. For pan-cytokeratin staining, the antigen retrieval was performed by pre-treating the tissue with Protease1 (from Roche) for 8 min while for other stainings, CC1 (Cell Conditioning 1 from Roche) was used for 60 min. All antibodies were incubated for 32 min Counterstaining was done with hematoxylin. Slides were scanned in an Aperio AT2 slide scanner (Leica) at magnification of 40X. The immunohistochemical expression was analyzed with Aperio ImageScope software (V12.4.0.7018). “Positive Pixel Count v9” algorithm was used for quantifying pan-cytokeratin expression. The total positive pixel was normalized to the total area of the annotated regions on tissue section (pixel/mm2). “Nuclear v9” algorithm was used for CD3 and CD8 positive cell count. The default set of parameters of the algorithms was modified according to the stain contrast and intensity of the scanned images.
10
Immunohistochemical Staining of Slides
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Slides were digested with Protease I (proprietary, Ventana Medical Systems Inc., Tucson, AZ) for 8 minutes and stained using a mouse monoclonal primary antibody (Ventana Medical Systems Inc., Tucson, AZ, 790–2988). OptiView DAB detection kit (proprietary, indirect, biotin free system, Ventana Medical Systems Inc., Tucson, AZ) was used for primary antibody detection.
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