Serial sections of 4 µm were prepared from paraffin-embedded tissue previously fixed in neutrally buffered formalin and mounted on 3-aminopropyltriethoxysilane-coated slides. After overnight exposition at 50 °C, tissue samples were de-paraffinized and rehydrated in graded alcohol and xylol. Heat-mediated antigen retrieval was performed utilizing a water bath and 0.01 M sodium citrate for 30 min at 97 °С. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 for 5 min and subsequent washing with PBS. Sections were blocked with 1% BSA/PBS and incubated overnight at 4 °C with 0.1 µg/ml of monospecific anti-CEACAM1 (clone C5-1X/8; LeukoCom), anti-CEACAM5 (clone 3E10-3; LeukoCom) and anti-CEACAM6 (clone 1H7-4B; LeukoCom), or an isotype control. After washing, sections were probed with biotinylated secondary rabbit anti-mouse antibody (Dako) for 1 h at room temperature. Sections were washed and incubated with VECTASTAIN ABC reagent (Vector Laboratories) for 30 min according to the Manufacturers´ protocol. Staining was visualized by diaminobenzidine (DAB) substrate controlling the developing color intensity via light microscopy. DAB-negative structures were identified by additional counterstaining with hematoxylin. Stained sections were mounted and documented by microscopy.
Vectastain abc reagent
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Canada, Norway
The Vectastain ABC reagent is a detection system designed for immunohistochemical labeling of target antigens in tissue sections. It consists of an avidin-biotin complex that amplifies the signal, enhancing the visualization of the immunoreaction.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Merck Group (13 mentions)
Biotinylated secondary antibody, by Vector Laboratories (11 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (7 mentions)
Biotinylated horse anti mouse igg, by Vector Laboratories (6 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (4 mentions)
Proteinase k, by Agilent Technologies (4 mentions)
Vectamount, by Vector Laboratories (4 mentions)
Entellan, by Merck Group (4 mentions)
Zen blue software, by Zeiss (4 mentions)
Normal goat serum (ngs), by Vector Laboratories (3 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (3 mentions)
Bx51 microscope, by Olympus (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Dab kit, by Vector Laboratories (3 mentions)
Biotinylated anti rabbit igg, by Vector Laboratories (3 mentions)
Leitz dm irb microscope, by Leica (3 mentions)
Vectastain abc kit, by Vector Laboratories (3 mentions)
Axio imager m2 imaging system, by Zeiss (3 mentions)
Vectastain universal secondary antibody, by Vector Laboratories (3 mentions)
Alcian blue, by Merck Group (3 mentions)
167 protocols using vectastain abc reagent
1
CEACAM Immunohistochemical Staining
2
Immunohistochemical Analysis of ISG15 and ZFP36
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Samples were paraffinized, rehydrated, and blocked with 3% of H2O2, followed by incubation with normal goat serum (Vector Laboratories, Burlingame, CA, USA). After incubation with the primary antibody specific for ISG15 (1:200; Catalogue No: 15981-1-AP; ProteinTech) and ZFP36 (1:1000; Catalogue No: 12737-1-AP; ProteinTech) overnight at 4 °C, sections were washed with PBS and incubated with the biotinylated secondary antibody (Vector Laboratories), followed by incubation with Vectastain ABC Reagent (Vector Laboratories). The visualization signals were developed using diaminobenzidine (DAB, Vector Laboratories), and the slides were counterstained with hematoxylin. The images were captured using an Olympus BX60 microscope (Olympus, Japan). Immunoreactivity was scored based on a combination of both the percentage and intensity of positively stained tumor cells to generate an H-score. Staining intensity was divided into four categories as follows: no staining, 0; weak staining, 1; moderate staining, 2; strong staining, 3. H-score was determined according to the formula: (% of weak staining × 1).
3
BVDV Antigen Detection in Tissues
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For BVDV antigen detection, 5-μm-thick paraffin-embedded tissue sections were deparaffinized and hydrated through a graded alcohol series before heat-induced antigen retrieval in 10 mM sodium citrate buffer (pH 6) for 30 min. A primary anti-BVDV monoclonal antibody (DMAB28412; Creative Diagnostics, Shirley, NY, USA) was used according to the manufacturer’s instructions. Next, the tissue sections were stained with a biotinylated anti-mouse IgG antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 1 h at room temperature, washed, and incubated with the VECTASTAIN ABC Reagent (Vector Laboratories) for 30 min. After washing, the tissue sections were allowed to react with a peroxidase substrate solution (Vector), rinsed, counterstained, mounted, examined by light microscopy, and photographed. Negative control slides were prepared by staining with isotype-matched IgG at the same dilution as that used for the primary antibody.
4
Immunohistochemical Analysis of Synuclein Pathology
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Immunohistochemical staining was performed according to a standard immunoperoxidase protocol for free-floating sections using following antibodies: mouse anti-dopamine- and cAMP-regulated phosphoprotein (DARPP-32; 1:5000; BD Biosciences, USA), mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000; Sigma, USA), rat anti-human α-syn (aa 116-131 hα-syn; dilution and company please; 15G7, Enzo Life Sciences, Germany), rabbit anti-phosphorylated α-syn (pα-syn; 1:1000; Abcam, UK), and mouse anti-nitrated α-syn (nα-syn; 1:1000; Invitrogen, Zymed Laboratories, USA). In case of the visualization of partially proteinase-K (PK)-resistant human α-syn aggregates, tissue slices were pre-stained with haematoxylin and digested with PK before the incubation with antibodies against α-syn (Neumann et al., 2004 (link)). Biotinylated horse anti-mouse IgG, biotinylated goat anti-rat IgG, and biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, USA) were used as secondary antibodies. Vectastain ABC reagent (Vector Laboratories, USA) and 3,3′-diaminobenzidine were applied to visualize the immunohistochemical binding sites. Stained sections were mounted onto gelatin-coated slides, dehydrated and coverslipped with Entellan.
5
Immunohistochemical Analysis of STAT3 in Breast Cancer Cells
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MCF-7 and MCF-7/ADR cells (2×104 cells/well) were plated in 8-well chamber slides, incubated overnight at 37°C and treated with apigenin (80 µM) for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min and treated with 3% hydrogen peroxide (H2O2) in methanol for 20 min. The cells were washed with PBS, blocked with 5% BSA in PBS for 1 h and incubated with an anti-STAT3 primary antibody (1:100 dilution) for 24 h at 4°C. After the cells were rinsed with PBS, they were incubated with anti-rabbit biotin-conjugated secondary antibody for 1 h at room temperature. Finally, the cells were treated with Vectastain ABC reagent (Vector Laboratories, Inc. Burlingame, CA, USA) for 30 min at 4°C and stained with diaminobenzidine tetra chloride (DAB) and hematoxylin. The cells were mounted with mounting medium and subsequently analyzed by microscopy.
6
Immunohistochemical Analysis of OSCC Tissues
7
Chondrogenic Differentiation of Cells
8
Immunohistochemical Analysis of Immune Cells
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Tissue blocks were fixed in 10% formalin. After paraffin embedding, 3-μm sections were subjected to staining. For the cell number counts, 5 randomly selected sites at 400× magnification (high-power field) were evaluated by use of light microscopy. For immunohistochemistry of CD4, CD8, or FoxP3, the sections were deparaffinized in xylene and rehydrated before antigen retrieval by boiling in citrate buffer (0.01 M citrate containing 0.5% Tween 20, pH 6.0). The sections were incubated in 10% bovine serum albumin (BSA) in PBS at room temperature for 1 h and then stained with rat anti-CD4 antibody (4SM95, 1:500 dilution; eBioscience, San Diego, CA, USA), anti-CD8 antibody (4SM15, 1:500 dilution; eBioscience), or anti-FoxP3 antibody (FJK-16s, 1:200 dilution; Invitrogen, Waltham, MA, USA) overnight at 4 °C, followed by biotinylated anti-rat IgG antibody (1:500; Vector Laboratories, Burlingame, CA, USA) and Vectastain ABC reagent (Vector Laboratories) at room temperature for 60 min and 30 min, respectively. Finally, the sections were stained by the use of a DAB Peroxidase Substrate Kit (Vector Laboratories) before imaging. For detection of apoptotic cells, a TumorTACS in Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA) was used according to the manufacturer’s instructions.
9
Histological Evaluation of Renal Injury
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The kidneys were fixed in 4% paraformaldehyde (PFA), dehydrated, embedded in paraffin and cut into 5 μm-thick sections. Renal sections were stained with periodic acid-Schiff (PAS). Semiquantitative scoring of glomerular sclerosis was performed using a five-grade method described previously51 (link). At least 50 glomeruli per section were evaluated by an examiner masked to the experimental conditions. For immunohistochemistry, paraffin-embedded sections were stained with primary antibodies against WT1 (ab89901, Abcam, Cambridge, MA, USA), MCP-1 (ab25124, Abcam, Cambridge, MA, USA), SERCA2 (ab2861, Abcam, Cambridge, MA, USA), and TNF-α (sc-52746, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. After incubation with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA), the sections were incubated with VECTASTAIN ABC reagent (Vector Laboratories, Burlingame, CA, USA) and color development was achieved using 3, 3′ diaminobenzidine (Vector Laboratories, Burlingame, CA, USA).
10
Glycoprotein Analysis of Parasitic Lysates
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The parasite lysates were treated with or without peptide N-glycosidase (PNGase) F (Roche Diagnostics, Basel, Switzerland), separated on SDS–PAGE gels, and transferred onto BioTrace PVDF membranes (PALL, Port Washington, NY). The membranes were blocked for 1 h with the protein-free PVDF Blocking Reagent (TOYOBO, Osaka, Japan). For lectin blotting, the membranes were incubated with 2 μg/mL of biotin-conjugated tomato lectin (Vector Laboratories) in 10 mM Tris HCl (pH 7.5) with 150 mM NaCl and 0.05% Tween20 (TBST); washed thrice with TBST; incubated for 30 min with Vectastain ABC reagent (Vector Laboratories); washed thrice with TBST; and developed with an enhanced chemiluminescent reagent (Promega, Fitchburg, WI), according to the manufacturer's instructions. For Western blotting, the antigen was detected with a 1:2000 dilution of polyclonal antisera in CanGetSignal reagent I (TOYOBO), followed by an HRP-conjugated anti-rabbit IgG (Promega) diluted in 1:4000 in CanGetSignal reagent II (TOYOBO).
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