Tumors were excised, frozen in OCT and stored in −80 °C. 10 μm cryo-sections were done and stained for F4/80. Sections were washed several times in PBS, and blocked with a PBS/0.4% Triton X-100/0.5% TSA (Tyramide Signal Amplification, Perkin Elmer Life Sciences, Boston, MA) blocking reagent/10% normal horse serum for 1 hour. Sections were then serially incubated with a rabbit anti-mouse F4/80 antibody (1:500; cat# 122604, Biolegend San Diego, CA) at 4°C for 24 h, and then washed in PBS six times for 5 min each wash. Sections were incubated at room temperature for 1 hour in biotinylated antibody (1:400, Vector Labs, Burlingame, CA) in a PBS/0.4% TSA blocking reagent/10% horse serum solution, and then with streptavidin-horseradish peroxidase diluted 1:100 in PBS/0.5% TSA blocking reagent. Subsequently, the sections were incubated with TSA fluorescein reagents diluted 1:50 in amplification diluent (Perkin Elmer Life Sciences) for 2 min. Sections were then washed and mounted onto glass slides. Sections were visualized using a fluorescent microscope by an investigator who was blinded to the sample source.
Biotinylated antibody
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated antibodies are antibodies that have been chemically modified to include a biotin molecule. Biotin is a small vitamin molecule that can bind strongly to the protein streptavidin. This allows biotinylated antibodies to be easily detected or captured using streptavidin-based detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Amplification diluent, by PerkinElmer (1 mentions)
Tyramide signal amplification, by PerkinElmer (1 mentions)
Rabbit anti mouse f4 80 antibody, by BioLegend (1 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Dojindo Laboratories (1 mentions)
Non immune rabbit igg, by Vector Laboratories (1 mentions)
Anti nitrotyrosine antibody, by Merck Group (1 mentions)
Non immune goat serum, by Vector Laboratories (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Goat or horse serum, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine dab substrate kit, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ab92552, by Abcam (1 mentions)
Blocking serum, by Vector Laboratories (1 mentions)
Ab213203, by Abcam (1 mentions)
Ab90716, by Abcam (1 mentions)
Abc kit protocol, by Vector Laboratories (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Histovt one, by Nacalai Tesque (1 mentions)
14 protocols using biotinylated antibody
1
Immunofluorescent Staining of Tumor Macrophages
2
ALP and Nitrotyrosine Immunohistochemistry
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Tissue sections were stained with hematoxylin and eosin. For ALP activity staining, the sections were stained with 0.1% naphthol AS-MX phosphate (Sigma-Aldrich) and 0.1% fast blue BB salt (Sigma-Aldrich) in 0.1 M Tris-buffer (pH 9.2) at 37 °C for 30 min14 (link). Immunohistochemistry with anti-nitrotyrosine antibody was performed as described previously14 (link). The sections were quenched with 0.3% H2O2 in PBS for 60 min and blocked with 10% non-immune goat serum (Vector Laboratories) for 30 min. Then, the samples were incubated with anti-nitrotyrosine antibody (1:100; Millipore) at 4 °C overnight. Next, the sections were incubated with a biotinylated antibody (1:200; Vector Laboratories) for 45 min, followed by avidin-biotin complex (1:100; Vector Laboratories) for 60 min. The sections were reacted with 0.02% 3,3″-diaminobenzidine tetrahydrochloride (Dojindo Laboratories) and 0.06% H2O2 in 0.05 M Tris buffer, pH 7.6 and counter-stained with hematoxylin. Immunohistochemical controls were incubated with non-immune rabbit IgG (Vector Laboratories) instead of the primary antibody. The sections were observed under an Axio Imager M2 microscope.
3
Immunohistochemical Staining of Paraffin-Embedded Tissue
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Prior to staining procedures, paraffin-embedded tissue sections were deparaffinized and hydrated to 70% ethanol. Tissues were stained with H&E staining following the manufacturer’s standard protocol. For immunohistochemistry, fixed tissue sections were permeabilized with 1% BSA in 0.1% Triton-X 100 (Sigma)/PBS for 10 min, inhibited endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol for 30 min, and blocked non-specific binding sites with 10% goat or horse serum (Vector Laboratories Inc., Burlingame, CA, United States) for 1 h. Primary antibodies listed in Supplementary Table 1 were used and incubated overnight at 4°C. Stained tissues were incubated with a biotinylated antibody at 1:100 (Vector Laboratories Inc., Burlingame, CA, United States) for 1 h, washed and treated with the Vectastain ABC kit and 3, 3′-diaminobenzidine (DAB) substrate kit according to manufacturer’s protocol (Vector Laboratories Inc., Burlingame, CA, United States).
4
Immunohistochemical Analysis of Lung Tissue
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Paraffin blocks were sliced to a thickness of 4 μm and attached to a silane-coated slide (Muto Pure Chemicals Co., Ltd., Japan). Each procedure was performed according to the ABC kit protocol (Vector Laboratories, USA). After deparaffinization and rehydration, slides were boiled in 0.1 M sodium citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. After cooling the slides to room temperature, 3% H2O2 in methanol was used to block the endogenous peroxidase activity. To suppress non-specific reactions, blocking serum (Vector Laboratories) was applied to the tissue. PCNA antibody (Abcam, ab92552, diluted 1:200), prosurfactant protein C (SPC, Abcam, ab90716, diluted 1:1000), and ubiquitin antibody (CC10, Abcam, ab213203, diluted 1:4000) were used as the primary antibodies. Biotinylated antibody (Vector Laboratories, USA) was used as a secondary antibody. It was then detected using the DAB Peroxidase Substrate Kit (Vector Laboratories, USA). All the slides were counterstained with hematoxylin (Gill III hematoxylin, Thermo, USA).
5
Immunostaining Protocols for Fish Gonads
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IF (immunofluorescence) and IHC (immunohistochemical) staining were performed as previously described [28 (link)]. The fish gonads were fixed with 4% paraformaldehyde in PBS at 4°C for 16 hours. The rehydrated slides were treated with HistoVT One (Nacalai tesque, Kyoto, Japan) to expose the antigens of the target protein. Anti-Vasa (1:1000 dilution; our own antibody) [28 (link)], anti-Proliferating cell nuclear antigen (Pcna; 1:250 dilution; product no. sc-7907; Santa Cruz Biotechnology, Billerica, MA, USA), anti-Brdu (1:1000 dilution; product no. MAB4072; Merk Millipore), and anti-Amh (1:1000; present study) were used for IHC and IF staining. For IHC staining, each section was rehydrated in PBS and incubated with 3% H2O2 in PBS. The section was then incubated with 5% nonfat milk powder for 30 min with antibody overnight at 4°C. This was followed by incubation with an appropriate biotinylated antibody (Vector Laboratories Inc., Burlingame, CA). Color formation was amplified with an ABC kit (avidin-biotin, Vector, Burlingame, CA, USA) and DAB (3,3’-diaminobenzidine, Sigma). For IF staining, the section was then incubated with 5% nonfat milk powder for 30 min with antibody overnight at 4°C. Alexa Fluor secondary antibodies (Invitrogen, Carlsbad, CA, USA) were used. All staining was conducted with triplicate sections for each tissue (n > 3 fish in each group).
6
Immunohistochemical Analysis of FFPE Brain Tissue
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Formalin-Fixed Para n-Embedded (FFPE) patient brain tissue sections were rst depara nized and rehydrated. Sections were quenched with 1% H 2 O 2 followed by antigen retrieval. Permeabilization was done using 0.1% Triton-X in 1X PBS and later-on blocked with 1% BSA, 3% Goat or Rabbit Serum, 0.05% Triton-X in 1X PBS. Sections were incubated overnight with primary antibodies, goat anti-Coronin 1A (Abcam, Cambridge, UK, 1:25), rabbit anti-GFAP (Dako, USA, 1:500) in blocking solution. Sections were washed 4 times with 0.02% Tween 20 in 1X PBS (PBST). Secondary antibody incubation was done for 2 hours with appropriate biotinylated antibody (Vector labs, USA, 1:500). Washing was done 4 times using PBST and sections were then treated with a complex of Avidin-Biotinylated HRP (Vector Labs, USA, 1:1) for 2 hours followed by 4 PBST washes. Sections were developed for 1-3 minutes using ImmPACT NovaRED kit (Vector Labs, USA) following the manufacturer's protocol, counterstained with Hematoxylin, and washed under running water. The sections were dried, treated with xylene, and then mounted using DPX mountant solution. Each section was imaged thrice randomly using Leica DMRXA2 (Leica, Wetzlar, Germany) microscope at 10x, 20x, and 40x magni cations. Images were then processed and analyzed using ImageJ software (NIH, USA).
7
Drosophila Embryonic Phenotypic Analysis
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Embryos were collected from grape-juice agar plates, dechorionated, devitellinized and fixed using standard methods. For each phenotypic analysis, at least 30 to 50 homozygous mutant embryos were analyzed by using a Leica TCS SP2 confocal microscope. The following primary antibodies were used: guinea pig anti-β3-Tubulin (1:10,000; Buttgereit et al., 1996 (link); Leiss et al., 1988 (link)), rabbit anti-Dmef2 kindly provided by Hanh Nguyen (Erlangen University, Germany) (1:500), rabbit anti-Myc (1:2000, cat. no 05-724, Merck Millipore Darmstadt, Germany), rabbit anti-GFP (1:1000, ab6556, Abcam, Cambridge, UK), rat anti-N-cadherin DN-Ex#8 from Hybridoma Bank (1:500), and rabbit anti-β-galactosidase (1:5000, Cappel Research Products Durham, NC). As secondary antibodies, we used biotinylated antibodies from Vector Laboratories (Peterborough, UK) for DAB staining and Cy2- and Cy3-conjugated secondary antibodies from Dianova GmbH (Hamburg, Germany).
8
Aggrecan Neoepitope Detection
9
Monocyte Differentiation and Characterization
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Human monocyte colony-stimulated factor (M-CSF) was a gift from Morinaga Milk Industry (Kanagawa, Japan). Lipopolysaccharide (LPS) was from Sigma-Aldrich (MO, USA). Granulocyte-monocyte colony-stimulated factor (GM-CSF), interferon-γ (IFN-γ), and interleukin-4 (IL-4) were from R&D system (MN, USA). Carboxyfluorescein diacetate succinimidyl ester (CFSE) and Hoechst 33342 were from Molecular Probe (OR, USA).
Sources of antibodies were as followed; anti–CD14-phycoerythrin (PE) (HCD14), anti–CD86-PE (IT2.2), anti–CD163-PE (GHI/61), anti–CD172a-PE (SIRPα/β) (SE5A5), anti–CD206-fluorescein isothiocyanate (FITC) (MMR, 15-2), anti–HLA-DR-FITC (L243), anti-F4/80 (CI: A3-1), and anti-CD172a (SE5A5) were from Biolegend (CA, USA); anti–CD47-FITC (2D3) and anti-CD47 (2D3) was from eBioscience (CA, USA); anti-CK19 (HPA002465) was from Sigma-Aldrich; Human Fc receptor blocking reagent was from Miltenyi Biotec (Bergisch Gladbach, Germany), isotype control antibodies were from Biolegend and DakoCytomation (Glostrup, Denmark), and biotinylated antibodies were from Vector Laboratories (CA, USA).
Sources of antibodies were as followed; anti–CD14-phycoerythrin (PE) (HCD14), anti–CD86-PE (IT2.2), anti–CD163-PE (GHI/61), anti–CD172a-PE (SIRPα/β) (SE5A5), anti–CD206-fluorescein isothiocyanate (FITC) (MMR, 15-2), anti–HLA-DR-FITC (L243), anti-F4/80 (CI: A3-1), and anti-CD172a (SE5A5) were from Biolegend (CA, USA); anti–CD47-FITC (2D3) and anti-CD47 (2D3) was from eBioscience (CA, USA); anti-CK19 (HPA002465) was from Sigma-Aldrich; Human Fc receptor blocking reagent was from Miltenyi Biotec (Bergisch Gladbach, Germany), isotype control antibodies were from Biolegend and DakoCytomation (Glostrup, Denmark), and biotinylated antibodies were from Vector Laboratories (CA, USA).
10
Immunohistochemical Analysis of Vascular Markers
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