Half of the tumours embedded in TissueTec were sectioned (10μm) using a cryostat (Leica) and fixated with acetone. The unconjugated rat anti-mouse B220-CD45r, CD3, CD4, and CD8 antibodies (hybridoma cell lines) were added for 1h at RT. Then, peroxidase blocker and biotinylated anti-rat Ig (polyclonal; BD) were added for 30min at RT. These biotinylated antibodies were detected with HRP labelled streptavidin (30min) and DAB substrate (BD). The tumour sections were dehydrated in 50%, 70%, 96%, and 100% ethanol (5min) and analysed using the Carl Zeiss Axioskop 40 microscope.
Dab substrate
Manufactured by BD
Sourced in United States
DAB substrate is a chromogenic substrate used for the detection and visualization of enzyme-labeled target molecules in various immunohistochemical and in situ hybridization applications. It produces a brown colored reaction product at the site of the target, enabling its localization.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop 40 microscope, by Zeiss (1 mentions)
Cryostat, by Leica camera (1 mentions)
Rabbit anti mouse ki67, by Abcam (1 mentions)
Upright microscope, by Olympus (1 mentions)
Hydroxyproline assay kit, by Chondrex (1 mentions)
Thioacetamide, by Merck Group (1 mentions)
Fl83b, by American Type Culture Collection (1 mentions)
Anti alpha smooth muscle actin antibody, by Abcam (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Goat anti rabbit igg hrp, by Abcam (1 mentions)
L glutamine 200 mm, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Plasmid dna purification kit, by iNtRON Biotechnology (1 mentions)
Abc solution, by Vector Laboratories (1 mentions)
Biotinylated anti mouse igg secondary antibody, by Vector Laboratories (1 mentions)
Vu4h5, by Santa Cruz Biotechnology (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Anti fibronectin, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
5 protocols using dab substrate
1
Immune Cell Profiling of Tumor Sections
2
Histological and Immunohistochemical Analysis of Skin Tissue
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Mice were euthanized after treatment, and approximately 1 cm2 of dorsal skin was excised and fixed in a buffered 4% paraformaldehyde solution. The tissue was then embedded in paraffin, sectioned at a thickness of 5 μm, and subjected to hematoxylin-eosin staining for histological examination. For immunohistochemical (IHC) analysis, sections were incubated with primary antibodies: rabbit anti-mouse Ki67 (1:500; Abcam, United States) and rabbit anti-mouse interleukin (IL)-17 (1:200; Abcam, United States). This was followed by application of a secondary antibody reagent kit (Maxim; Fuzhou, China) and visualization using DAB substrate (BD Pharmingen, United States). After counterstaining with hematoxylin, the sections were dehydrated, cleared, and mounted on glass slides for observation and measurement under an upright microscope (Olympus Corporation, Japan).
3
Thioacetamide-Induced Liver Fibrosis Study
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Thioacetamide was purchased from Sigma–Aldrich, USA. DAB substrate and DAB staining solution were purchased from BD Pharmingen (San Diego, CA 92121, USA). The HepG2 and FL83B cell lines were purchased from ATCC (Manassas, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo Scientific, USA. L-Glutamine (200 mM) was purchased from Sigma–Aldrich, USA. A hydroxyproline assay kit was purchased from Chondrex (WA, USA). Anti-alpha smooth muscle actin antibody-Lot:GR283004-24 (Abcam, Cambridge, UK), goat anti-rabbit IgG H&L (Alexa Fluor1 488) Lot:GR306624-1 (Abcam, Cambridge, UK), hepatitis B core antigen Lot:#SF2406841H (Invitrogen, Thermo Fisher Scientific, South Korea), and goat anti-rabbit IgG (HRP) Lot:GR247075-7 (Abcam, Cambridge, UK) were used. A plasmid DNA purification kit was purchased from Intron Biotechnology (Lynnwood WA). The DNA isolation kit was purchased from QIAamp (DNA Mini kit, Germany). An ELISA kit for HBV antigen analysis was purchased from Wanti-Biopharm (Beijing). All other reagents used were of analytical or chromatographic grade.
4
Immunohistochemistry of Underglycosylated MUC1
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IHC was performed as described in Cascio et al. (48 (link)). Briefly, deparaffinized and rehydrated 4 μm sections of tumor specimen were incubated for 15 min at RT in a 30%H2O2/methanol solution (1:10) to block endogenous peroxidase activity. Slide were washed 3 times with PBS 1X, antigens were retrieved in 0.1% citrate buffer pH 6 for 5'at 120°C. Sections were permeabilized in PBS-0.2% Tween20 (5'at RT)and incubated with incubated PBS-2%BSA (20' at RT) to block non-specific binding. Samples were then incubated 1 h RT with a 1:40 dilution in PBS−2% BSA of the anti Mucin 1 antibody that specifically recognizes the underglycosylated, tumor specific form, of MUC1 (VU-4H5, Santa Cruz Biotechnology). Slides were washed in PBS-0.2% TWEEN20 (5'at RT) and incubated for 1 h with the biotinylated anti-mouse IgG secondary antibody (Vector Laboratories dilution 1:200 in PBS-BSA2%) and washed in PBS-0.2% TWEEN 20 for 5'at RT. Slices were incubated with ABC solution (Vector Laboratories) for 30'at RT washed, developed with DAB substrate (BD Pharmingen).
5
Quantifying Renal Extracellular Matrix Proteins
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Five-micrometer sections were prepared from a paraffin-embedded kidney for immunohistochemistry as described previously (12 (link)). Briefly, kidney sections were blocked with normal goat serum and the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer protocol. To quantify the expression levels of renal collagen IV, fibronectin, and LRP6, sections were incubated separately with anti–collagen IV (Millipore, Billerica, MA), anti-fibronectin (Santa Cruz Biotechnology, Dallas, TX), anti–β-catenin (Santa Cruz Biotechnology), anti-PPARα (Abcam, Eugene, OR), and anti-LRP6 (in-house antibody) (17 (link)) antibodies. ABC reagent (Vector Laboratories) was used, and the color was developed by DAB substrate (BD Biosciences, San Jose, CA) according to manufacturer instructions.
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