Four micron paraffin sections of mouse kidney were stained with goat antibody to collagen III alpha 1/COL3A1 (Novus Biologicals #NBP1-26547) using a three-step avidin-biotin horseradish peroxidase method (Vector Labs, Newark, CA, USA). Briefly, sections were deparaffinized, rehydrated and antigen retrieval was performed using 10 mM sodium citrate, pH 6.0 buffer for 10 min using an electric pressure cooker. After cooling, sections were blocked with 3% hydrogen peroxide and incubated overnight at 4 °C with 10% normal rabbit serum. Primary antibody was incubated for one hour at room temperature, followed by blocking for endogenous biotin using the Biocare Medical Avidin Biotin kit (#AB972H). Secondary and tertiary steps were for 30 min each and labeling for collagen 3 was detected using the SignalStain DAB Substrate kit (Cell Signaling #8059). Kidney sections stained for Collagen 3 were scanned using the Leica Aperio CS2 Scanscope at 20× and staining quantification was performed using Aperio Imagescope. Specifically for quantification, 27–31 separate cortical regions from each section were analyzed using the Positive Pixel Count v9 algorithm (5 animals per group), giving the ratio of total Collagen 3 positively stained pixels to the total number (stained and unstained) pixels in each region.
Signalstain dab substrate kit
Manufactured by Cell Signaling Technology
Sourced in United States
The SignalStain DAB Substrate Kit is a laboratory product designed for the detection and visualization of target proteins in immunohistochemistry and immunocytochemistry applications. It contains the necessary components to facilitate the colorimetric development of the DAB (3,3'-Diaminobenzidine) chromogenic reaction.
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Lab products found in correlation
Signalstain boost ihc detection reagent, by Cell Signaling Technology (11 mentions)
Signalstain boost detection reagent, by Cell Signaling Technology (6 mentions)
Anti ki67 antibody, by Abcam (2 mentions)
β catenin antibody, by Cell Signaling Technology (2 mentions)
Hyclone bovine serum albumin, by GE Healthcare (2 mentions)
Acrytol mounting medium, by Electron Microscopy Sciences (2 mentions)
Bloxall, by Vector Laboratories (2 mentions)
Decloaking chamber, by Biocare Medical (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (2 mentions)
Tunel in situ cell death detection kit, by Roche (2 mentions)
Ab 3014, by Cell Signaling Technology (2 mentions)
Ez retriever system, by BioGenex (2 mentions)
Anti β catenin antibody, by Abcam (2 mentions)
Goat anti rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Goat serum, by Cell Signaling Technology (2 mentions)
Biotinylated goat anti rabbit igg antibody, by Vector Laboratories (2 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
Ki 67 antibody, by Cell Signaling Technology (2 mentions)
62 protocols using signalstain dab substrate kit
1
Quantifying Collagen III in Mouse Kidney
2
In Situ Hybridization and Immunohistochemistry Techniques
3
Characterization of Stemness and EMT in Cancer Cells
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Iscove’s Modified Dulbecco’s Medium (IMDM), fetal bovine serum (FBS), Lipofectamine LTX, plus reagent, matrigel, DMEM/F12, B27 supplement, N2 supplement, and Anti-Neu2 were from Invitrogen (MA5-25555) (USA), as well as CD133 and CD44 (BD phermingen) and Oct-4A(2840), Sox2(3579), Nanog(4903), Slug(9585S), Snail(3879S), Cyclin D1(2978S), mTOR(2983), phospho-mTOR (Ser2481)(2974), Rictor(9476), GSK-3β(9315), Gli1(3538S), Sufu(2522S),GSK-3β(9315), Phospho-GSK-3β-Ser9(5558), Akt(4691), Phospho-Akt-Ser473(4060), β-Actin(4970), HRP-conjugated anti-rabbit antibodies(7047S) and anti-mouse secondary antibodies (7076). SignalStain® DAB Substrate Kit(8059) was from Cell Signaling Technology (Danvers, USA). Shh(sc-9024) antibody was from Santa Cruz Biotechnology (Texas, USA). Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), anti-Caspase3, active antibody(C8487) and other chemicals were from Sigma–Aldrich. RevertAid First Strand cDNA Synthesis Kit (K1622) and Maxima SYBR Green qPCR Master Mix (2X) were from Thermo Scientific, USA (K0251); biotinylated Sambucus nigra agglutinin (SNA)(B-1305) and biotinylated Maackia amurensis agglutinin (MALII) (B-1265) were from Vector Laboratories (USA).Rictor plasmid Addgene plasmid #11367) was from Addgene and PcDNA3.1-Neu2 was a kind gift from Dr. Eugenio Monti.
4
Immunohistochemical Detection of NKp46
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Tissue sections (5 μm) were deparaffinized in xylenes and were rehydrated by passing through serial dilutions of ethanol in distilled water. Heat-induced antigen retrieval was performed in ImmunoActive antigen retrieval solution (Matsunami Glass Ind. Ltd.) with microwave thrice for 5 min. Goat anti-mouse NKp46 antibody (R&D systems) was applied to the sections; then, the sections were incubated overnight at 4°C. After washing with phosphate-buffered saline (PBS), the sections were incubated with goat IgG horseradish peroxidase (HRP) polymer antibody (R&D systems). HRP reacted with the 3, 3’-diaminobenzidine (DAB) substrate using the SignalStain® DAB Substrate kit (Cell Signaling Technology). The sections were counterstained with hematoxylin.
5
Immunohistochemical Analysis of LDH-A in Murine Pancreatic Tissue
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Paraformaldehyde-fixed, paraffin-embedded murine pancreatic tissue sections were deparaffinized and rehydrated. For antigen retrieval, tissue was boiled with 1X citrate buffer, pH 6.0 (Sigma), for 15 min. We then put slides in 3% H2O2 for 15 min to block endogenous peroxidases. Nonspecific epitopes were blocked with HyClone Bovine Serum Albumin (GE Healthcare Life Sciences) for 15 min. The sections were incubated overnight at 4 °C with LDH-A Antibody (Cell Signaling Technology). This was followed by an HRP-conjugated secondary antibody for 1 h. Then we applied Signal Stain DAB Substrate Kit (Cell signaling) following the manufacturer’s instructions. Finally, slides were counterstained with hematoxylin and mounted in Acrytol Mounting Medium (Electron Microscopy Sciences).
6
Dual Immunohistochemical Staining Protocol
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FFPE sections (thickness of 4 μm) were deparaffinized in xylene and rehydrated in ethanol. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide in methanol. Double staining was performed using the SignalStain IHC Dual Staining Kit (#36084, Cell Signaling Technology, Danvers, MA, USA). Slides were incubated with a primary mouse monoclonal anti-LAMP-3 antibody (clone 16H11.2, #3503452, Merck, Darmstadt, Germany; 1:500) at 4 °C overnight. Sections were then incubated at room temperature for 30 min with a secondary horseradish peroxidase (HRP)-conjugated mouse antibody (SignalStain Boost IHC Detection Reagent, Cell Signaling Technology). Visualization was performed with diaminobenzidine (SignalStain DAB Substrate Kit, Cell Signaling Technology). Antigens were retrieved by autoclaving for 10 min in Target Retrieval Solution (Agilent Technologies, Santa Clara, CA, USA) (100 °C, pH 9.0) for TIM-3. Slides were then incubated with a primary rabbit monoclonal anti-TIM-3 antibody (D5D5R XP, #45208, Cell Signaling Technology; 1:200) at 4 °C overnight. Slides were treated with SignalStain Boost IHC Detection Reagent (AP, Rabbit) (Cell Signaling Technology), and visualization was performed with Vibrant Red (SignalStain Vibrant Red Alkaline Phosphatase Substrate Kit, Cell Signaling Technology). Counterstaining was conducted with hematoxylin.
7
Immunohistochemical and Immunofluorescent Staining
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Sections were de-paraffinized and hydrated. Antigen retrieval was performed in a decloaking chamber (Biocare Medical, Pacheco, CA, USA) using citrate buffer pH 6.0. Endogenous peroxidases and non-specific binding sites were blocked with Bloxall (Vector Laboratories, Burlingame, CA, USA), rodent block (Biocare Medical) and horse serum (10%). Primary antibodies anti-NR2F6/Ear2 (ab137496, Abcam, Waltham, MA, USA, 1:3000 dilution), anti-GPNMB (ab188222, Abcam, Waltham, MA, USA, 1:1000 dilution) were added overnight. An HRP-conjugated horse anti-rabbit IgG Polymer (Vector Laboratories) was used as secondary for 30 min and the signal was developed using the SignalStain DAB Substrate Kit (Cell Signaling). For IF, anti-CD68 (ab283654, Abcam, Waltham, MA, USA, 1:1000 dilution) and anti-CD72 (AF1279, R&D Systems, Minneapolis, MN, USA, 1:40 dilution) antibodies were coadministered overnight and were labeled with horse anti-rabbit or anti-goat Dylight 488 and 594 (Vector Laboratories, 1:300 dilution) secondary antibodies for 30 min. Alternatively, an Opal kit (Akoya Biosciences, Marlborough, MA, USA) was used with anti-NR2F6/Ear2 (ab137496, Abcam, Waltham, MA, USA, 1:700 dilution) and anti-CD206/Mrc1 (24595T, Cell Signaling, Danvers, MA, USA, 1:500 dilution) primary antibodies.
8
Immunohistochemical Analysis of Tumor Markers
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Tumors were harvested from MMTV-PyMT transgenic mice at the end point of treatment. Paraffinized tumor sections were permeabilized in 0.2% Triton X-100 after de-paraffinized in xylene and rehydrated in ethanol. Tumor sections were immersed in sodium-citrate buffer (10 mM, pH 6.0) for antigen retrieval and blocked in 5% normal goat serum (Cell Signaling Technology, Danvers, MA). The slides were then incubated with diluted primary antibodies Bax, MMP-7, PTEN, and β-catenin (Cell Signaling Technology, Danvers, MA) at 4 °C overnight. After washing, the slides were incubated with SignalStain Boost Detection Reagent (Cell Signaling Technology, Danvers, MA) and developed color according to SignalStain DAB Substrate Kit (Cell Signaling Technology, Danvers, MA) manufacturer’s guidelines. The study was conducted in accordance with the guidelines approved by Department of Health (Hong Kong, Ethic approval, 15–322). The images were detected by Leica DME microscope (Meyer Instruments, Inc., Houston, USA) and analyzed by ScopeImage 9.0 Image-Processing Software (BP Integrated Technologies, Inc., Calamba, PH).
9
Immunohistochemical and Immunofluorescence Tissue Staining
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Tissues were fixed in 10% formalin, embedded, and sectioned. The samples were fully deparaffinized in 100% xylene and rehydrated in ethanol at gradually lower concentrations. The slices were then soaked in citrate buffer (pH 6.0) and boiled in Retriever (Electron Microscopy Sciences, PA, USA) for antigen retrieval using standard procedures. For immunohistochemical (IHC) staining, the slices were stained with primary antibodies, followed by signal detection using a Signal Stain DAB Substrate Kit (Cell Signaling Technology, MA, USA). For immunofluorescence, the tissue samples or cells were fixed with 4% paraformaldehyde at room temperature and stained with primary antibodies, as described above. The general staining procedure was based on the standard protocols. Images were acquired using a BX53 microscope (Olympus, Japan).
10
Recombinant Human TNF-α Signaling Pathway
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Recombinant human TNF-α was obtained from Cayman (Ann Arbor, MI, USA). Lipofectamine 2000 and Trizol reagents were purchased from Invitrogen (Carlsbad, CA, USA). The nucleoprotein and cytoplasm-protein extraction kits and ready-to-use immunohistochemical SP kits were purchased from KeyGEN BioTECH (Nanjing, JiangSu, China). The SignalStain DAB substrate kit was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting AKT, β-catenin and all unconjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies targeting phospho-AKT (p-AKT) and E-cadherin were purchased from Epitomics (Burlingame, CA, USA). Anti-phospho-PKD2 antibodies (for the IHC Assay) were purchased from Sigma. Antibodies against N-cadherin, vimentin, Zo-1, phospho-GSK3β (p-GSK3β), PKD1, PKD2, PKD3, phospho-S6K1(p-S6K1), phospho-ERK1/2 (p-ERK1/2), β-actin and histones were obtained from Cell Signaling Technology. Antibodies targeting green fluorescent protein (GFP) were purchased from Abcam (Cambridge, MA, USA). Alexa-488- and 594-conjugated secondary antibodies were purchased from Molecular Probes (Invitrogen).
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