Tumors (n = 2/time-point) were sampled at days 1, 3, and 7 after intravenous injection of intact 059-053 (25 µg) into tumor-bearing mice, fixed in 10% (v/v) neutral buffered formalin, and embedded in paraffin for sectioning. Untreated tumors were used as a control. Sections (1-µm thick) were immunostained with a goat anti-EMMPRIN/CD147 antibody (AF972, R&D Systems, Minneapolis, MN, USA, diluted 1:500) as described previously [15 (link)]. MMP2 and MMP9 were detected using anti-MMP2 and anti-MMP9 polyclonal antibodies, respectively, as the primary antibody (Sigma-Aldrich Japan, Tokyo, Japan; diluted 1:200 and 1:250, respectively). Nonspecific binding was blocked by Protein Block Serum-Free reagent (Agilent Technologies, Santa Clara, CA, USA). The specimens were incubated overnight at 4 °C with the primary antibody. The secondary antibody (Dako EnVision+ system, horseradish peroxidase-labeled polymer for anti-rabbit, Agilent Technologies (Santa Clara, CA, USA) was applied for 30 min at room temperature and then visualized with a diaminobenzidine staining reagent (Dako Liquid DAB+ Substrate Chromogen system, Agilent Technologies).
Dako liquid dab substrate chromogen system
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The Dako Liquid DAB+ Substrate Chromogen System is a laboratory equipment product designed for use in immunohistochemistry (IHC) and in situ hybridization (ISH) procedures. It provides a chromogenic substrate for the visualization of target antigens or nucleic acid sequences in tissue sections or cell preparations.
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Lab products found in correlation
Dako mayer s hematoxylin lillie s modification histological staining reagent, by Agilent Technologies (6 mentions)
Dako envision dual link system hrp, by Agilent Technologies (4 mentions)
Dako envision system, by Agilent Technologies (3 mentions)
Mayer s hematoxylin, by Agilent Technologies (3 mentions)
Dako automation hematoxylin histological staining reagent, by Agilent Technologies (3 mentions)
Anti rabbit hrp conjugated antibody, by Agilent Technologies (3 mentions)
Mayer s hematoxylin, by Merck Group (3 mentions)
Vectastain elite abc reagent, by Vector Laboratories (3 mentions)
Histofine simple stain rat max po, by Nichirei Biosciences (2 mentions)
Ix51 microscope, by Olympus (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti ki67, by Abcam (2 mentions)
Ab64377, by Abcam (2 mentions)
Anti goat ucp1 antibody sc 6528, by Santa Cruz Biotechnology (2 mentions)
Anti α actin, by Abcam (2 mentions)
Anti ephb4, by Santa Cruz Biotechnology (2 mentions)
Anti cd34, by R&D Systems (2 mentions)
Anti cd68, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Envision system hrp labelled polymer anti rabbit, by Agilent Technologies (2 mentions)
61 protocols using dako liquid dab substrate chromogen system
1
Immunohistochemical Analysis of Tumor Biomarkers
2
Immunohistochemical Evaluation of GGT Protein
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Expression of GGT protein was observed by immunohistochemical staining. Paraffin sections
were deparaffinized and subsequently rehydrated. The sections were activated with
Tris-EDTA (pH 9.0) at 98°C for 30 min and at room temperature for 30 min. Endogenous
peroxidase activity was blocked with Dako Rael Peroxidase-Blocking (Agilent, Santa Clara,
CA, USA) for 10 min at room temperature. Non-specific protein binding was blocked with 8%
skim milk for 40 min at 37°C. Then, sections were incubated with primary antibodies at 4°C
overnight. The primary antibody used was GGT1 antibody (3E6) sc-100746 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA, diluted at 1:500). The efficacy of the antibody for
canine GGT was confirmed using canine kidney tissue lysate which has much of GGT (data not
shown). The sections were visualized by using Dako EnVision + System (Agilent) and Dako
Liquid DAB + Substrate Chromogen System (Agilent). The sections were also counterstained
with hematoxylin.
GGT immunoresponse intensity was subjectively evaluated from (−) to (+++) based on the
previous study [7 (link)]. The scoring system used was: -,
no GGT immune response; +, rare to 10% of GGT-positive tumor cells; ++, 10% to less than
one-half of tumor cells were GGT-positive; +++, over one-half of the tumor cells were
GGT-positive.
were deparaffinized and subsequently rehydrated. The sections were activated with
Tris-EDTA (pH 9.0) at 98°C for 30 min and at room temperature for 30 min. Endogenous
peroxidase activity was blocked with Dako Rael Peroxidase-Blocking (Agilent, Santa Clara,
CA, USA) for 10 min at room temperature. Non-specific protein binding was blocked with 8%
skim milk for 40 min at 37°C. Then, sections were incubated with primary antibodies at 4°C
overnight. The primary antibody used was GGT1 antibody (3E6) sc-100746 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA, diluted at 1:500). The efficacy of the antibody for
canine GGT was confirmed using canine kidney tissue lysate which has much of GGT (data not
shown). The sections were visualized by using Dako EnVision + System (Agilent) and Dako
Liquid DAB + Substrate Chromogen System (Agilent). The sections were also counterstained
with hematoxylin.
GGT immunoresponse intensity was subjectively evaluated from (−) to (+++) based on the
previous study [7 (link)]. The scoring system used was: -,
no GGT immune response; +, rare to 10% of GGT-positive tumor cells; ++, 10% to less than
one-half of tumor cells were GGT-positive; +++, over one-half of the tumor cells were
GGT-positive.
3
Histological Analysis of Mouse Eyes
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Eyes were removed from 22-week-old mice under general anaesthesia induced as mentioned previously. The eyes were fixed in 4% paraformaldehyde, after which the corneal buttons were excised, subjected to standard processing, embedded in paraffin, and sectioned at 5–7 μm. These sections were then deparaffinised using xylene, dehydrated using graded ethanol, and stained using H&E, Congo-red to detect the presence of amyloids and Masson’s trichrome to detect hyaline, as per the manufacturers’ instructions.
The deparaffinised sections were also evaluated using immunohistochemistry. The sections were washed with PBS, blocked with 5% skim milk for 30 min at room temperature, and incubated with a rabbit anti-TGFBI antibody (Proteintech Group Inc., Rosemont, IL;#10188-1-AP, 1:1000) for 60 min at room temperature. Adjacent sections were processed without primary antibody used as negative controls. Peroxidase-labelled anti-rabbit polyclonal antibody (Nichirei Biosciences Inc., Tokyo, Japan, # 714341) was used as the secondary antibody and the sections were incubated for 30 min at room temperature. Immunoreactivity was visualized using the Dako Liquid DAB + Substrate Chromogen System (Agilent Technologies, Inc., Santa Clara, CA, # K3467).
The deparaffinised sections were also evaluated using immunohistochemistry. The sections were washed with PBS, blocked with 5% skim milk for 30 min at room temperature, and incubated with a rabbit anti-TGFBI antibody (Proteintech Group Inc., Rosemont, IL;#10188-1-AP, 1:1000) for 60 min at room temperature. Adjacent sections were processed without primary antibody used as negative controls. Peroxidase-labelled anti-rabbit polyclonal antibody (Nichirei Biosciences Inc., Tokyo, Japan, # 714341) was used as the secondary antibody and the sections were incubated for 30 min at room temperature. Immunoreactivity was visualized using the Dako Liquid DAB + Substrate Chromogen System (Agilent Technologies, Inc., Santa Clara, CA, # K3467).
4
AaTV Persistence and DENV-1 Co-Infection
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A C6/36 cell culture persistently infected with AaTV was established following a previously established protocol, with modifications [30 (link)]. Briefly, a monolayer of C6/36 cells was inoculated with AaTV-infected supernatant in a 25 cm2 culture flask at 28 °C/5% CO2. The inoculum was removed after virus adsorption and cells were covered with fresh culture medium. AaTV-infected cells were sub-cultured weekly. Persistent AaTV infection was confirmed by RT-PCR and immunohistochemistry.
AaTV-infected C6/36 cells were seeded in a six-well plate at 28 °C/5% CO2 overnight. The cells were inoculated with the Dengue virus serotype 1 (DENV-1; D1/Hu/Saitama/NIID100/2014) with a multiplicity of infection (MOI) of 0.01 and 1. Cell supernatants were harvested on days 0, 1, 2, 5, 6, and 7 post-inoculation. The detection and quantification of DENV-1 were done by focus-forming assay using mAb4G2-antibody produced from D1-4G2-4-15 mouse hybridoma cells (ATCC HB-112, American Type Culture Collection), Dako EnVision+ System-HRP Labeled Polymer Antimouse, and DAKO Liquid DAB+ Substrate Chromogen System (Agilent Technologies, Santa Clara, CA), following the manufacturer’s instructions. The viral titers were plotted against time. The experiments were run in triplicates, and all experiments were run with controls.
AaTV-infected C6/36 cells were seeded in a six-well plate at 28 °C/5% CO2 overnight. The cells were inoculated with the Dengue virus serotype 1 (DENV-1; D1/Hu/Saitama/NIID100/2014) with a multiplicity of infection (MOI) of 0.01 and 1. Cell supernatants were harvested on days 0, 1, 2, 5, 6, and 7 post-inoculation. The detection and quantification of DENV-1 were done by focus-forming assay using mAb4G2-antibody produced from D1-4G2-4-15 mouse hybridoma cells (ATCC HB-112, American Type Culture Collection), Dako EnVision+ System-HRP Labeled Polymer Antimouse, and DAKO Liquid DAB+ Substrate Chromogen System (Agilent Technologies, Santa Clara, CA), following the manufacturer’s instructions. The viral titers were plotted against time. The experiments were run in triplicates, and all experiments were run with controls.
5
Histological Analysis of Islet Transplants
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Two weeks after islet transplantation, the scaffolds were removed, fixed in 10 % neutral-buffered formalin, and embedded in paraffin blocks. Four-micrometer-thick paraffin sections were cut and routinely stained with hematoxylin and eosin (HE) and Verhoeff-Van Gieson elastin stain. Immunohistochemical detection of insulin (mouse monoclonal, MU029-UC, Biogenex, USA) and luciferase (mouse monoclonal, Luci 21 1-107, Novus Biologicals, USA) was performed on 4-μm-thick paraffin sections. The primary anti-insulin antibody was detected using Simple Stain MAX PO (MULTI) Universal Immuno-peroxidase Polymer anti-mouse, anti-rabbit Histofine (Nichirei Biosciences, Japan). Histofine Simple Stain Rat MAX PO (Nichirei, Japan) was used for detecting luciferase. Finally, visualization was performed using the Dako Liquid DAB+ Substrate-Chromogen System (Agilent, USA) and counterstaining with Harris’s hematoxylin. The slides were viewed using standard light microscopy (Olympus BX41).
6
Immunohistochemical Analysis of Tissue Samples
7
Immunohistochemical Analysis of Renal Tissue
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Immunohistochemistry was performed as previously described [22 (link)]. For immunostaining, mouse tissues were fixed with Mildform (Wako Pure Chemical, Tokyo, Japan). For human renal tumor tissues, a prefixed human tissue array was purchased (KD2082a, US Biomax Inc., Rockville, MD, USA). After paraffinization, the tissues were sectioned and deparaffinized using xylene and ethanol, followed by antigen retrieval using Universal HIER Antigen Retrieval Reagent (Abcam). Samples were subjected to hematoxylin & eosin staining or immunostaining. After blocking with Block ACE (Bio‐Rad, Hercules, CA, USA), the samples were incubated with primary antibodies and the appropriate secondary antibodies (Table S4 ) and stained using a Dako Liquid DAB+ Substrate Chromogen System (Agilent Technologies, Santa Clara, CA, USA) and Mayor’s hematoxylin. Images were captured using an AX80 microscope (Olympus, Tokyo, Japan).
To detect apoptosis, In Situ Cell Death Detection Kit (TMR red; Roche Diagnostics) and DAPI Fluoromount‐G (Southern Biotech, Birmingham, AL, USA) were used as previously described [32 (link)]. Fluorescent images were captured using a BZ‐X710 microscope (KEYENCE, Osaka, Japan).
To detect apoptosis, In Situ Cell Death Detection Kit (TMR red; Roche Diagnostics) and DAPI Fluoromount‐G (Southern Biotech, Birmingham, AL, USA) were used as previously described [32 (link)]. Fluorescent images were captured using a BZ‐X710 microscope (KEYENCE, Osaka, Japan).
8
MMP13 Immunohistochemistry Protocol
9
Immunohistochemical Staining of Tissue Sections
10
Quantifying Arachidonic Acid in Liver Extracts
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Free ARA content in liver Triton X-100 extracts [16 (link)] was evaluated by capture ELISA. Wells were coated with 250 ng unlabelled rabbit polyclonal antibody to ARA (MyBioSource, San Diego, CA, USA, MBS2003715 ) overnight at 4°C. Following washing in 0.1 M PBS/0.05% Tween 20 (PBS-T), 200 μg liver protein of naive, control and immunized mice were added in duplicate wells to a total volume of 100 μL PBS-T, and incubated for 2 h at room temperature. The wells were thoroughly washed and added with 150 ng horseradish peroxidase-linked polyclonal antibody to ARA (MyBioSource, MBS2051576) for 1 h at room temperature. The reaction was visualized 30 min after adding 3,3’,5,5’ tetramethylbenzidine substrate (Sigma).
Arachidonic acid content was additionally evaluated by immunohistochemistry as described previously [8 (link),9 (link),17 (link)], except that liver sections were exposed to 3% hydrogen peroxide (Sigma) to block endogenous peroxidase activity, then incubated with 0 or 0.5 μg horseradish peroxidase-linked polyclonal antibody to ARA (MyBioSource, MBS2051576) overnight at 10°C. The reaction was visualized with Dako Liquid DAB + Substrate Chromogen System (Agilent Dako, Santa Clara, CA, USA). Photographs were acquired by light microscopy.
Arachidonic acid content was additionally evaluated by immunohistochemistry as described previously [8 (link),9 (link),17 (link)], except that liver sections were exposed to 3% hydrogen peroxide (Sigma) to block endogenous peroxidase activity, then incubated with 0 or 0.5 μg horseradish peroxidase-linked polyclonal antibody to ARA (MyBioSource, MBS2051576) overnight at 10°C. The reaction was visualized with Dako Liquid DAB + Substrate Chromogen System (Agilent Dako, Santa Clara, CA, USA). Photographs were acquired by light microscopy.
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