Sections from formalin-fixed, paraffin-embedded liver specimens were stained for PD-L1 using 1:50 diluted anti-mouse PD-L1 rabbit IgG (Ab58810; Abcam, Cambridge, UK), anti-rabbit EnVision System-HRP (DakoCytomation, Glostrup, Dennmark), and Diaminobenzidine substrate-chromogen (DAB; DakoCytomation). For CK-7 detection in cultured mouse cholangiocytes, cells seeded on coverslips were fixed with 2% paraformaldehyde and further permeabilized with 0.1% Triton X-100. Cell preparations were stained for 1 hour at 37°C with 1:400 diluted anti-CK-7 mouse mAb IgG1 (RCK105, Santa Cruz Biotech) and then washed 3 times with PBS containing 1% BSA. Respective horseradish peroxidase (HRP)-conjugated secondary antibody was used at 1:400 dilution during 1 hour at 37°C. After washing as above, cells were finally treated with DAB solution (DakoCytomation) 10 minutes at room temperature. Images were acquired in an Axio Imager M1 microscope (Zeiss).
Envision system hrp anti rabbit
Manufactured by Agilent Technologies
Sourced in Japan
The EnVision + System HRP anti rabbit is a laboratory equipment product from Agilent Technologies. It is a detection system designed for use with horseradish peroxidase (HRP) labeled secondary antibodies that target rabbit primary antibodies. The core function of this product is to facilitate the visualization and detection of target analytes in various immunoassay and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager m1 microscope, by Zeiss (1 mentions)
Dab solution, by Agilent Technologies (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Dual endogenous enzyme block, by Agilent Technologies (1 mentions)
Dab substrate chromogen system, by Agilent Technologies (1 mentions)
Blocking buffer, by Merck Group (1 mentions)
Bz x710, by Keyence (1 mentions)
Ab16669, by Abcam (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
α sma, by Thermo Fisher Scientific (1 mentions)
Proliferating cell nuclear antigen (pcna), by Agilent Technologies (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Safranin o, by Merck Group (1 mentions)
Pronase, by Merck Group (1 mentions)
Fast green, by Merck Group (1 mentions)
10 protocols using envision system hrp anti rabbit
1
PD-L1 and CK-7 Immunostaining Protocols
2
Immunohistochemistry of Mouse CD31 Protein
3
Immunohistochemical Analysis of CD3 and CD45
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Paraffin-embedded tumor specimens were cut into 5-μm sections, and tissue slides were deparaffinized with Histo-Clear (National Diagnostics, Atlanta, GA, USA) and rehydrated by sequential incubation in ethanol at different concentrations. Antigen retrieval was performed by incubation of the slides in boiling citrate buffer for 20 min, followed by incubation with 3% H2O2 and blocking buffer (Sigma-Aldrich, St. Louis, MO, USA). The slides were probed with rabbit anti-CD3 antibody [SP7] (ab16669; dilution 1:150; Abcam, Cambridge, UK) or rabbit anti-CD45 antibody [D3F8Q] (70257S; dilution 1:200, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The signal was amplified using Anti-Rabbit EnVision+ System, HRP (Dako, Carpinteria, CA, USA) for 1h at room temperature and visualized using the liquid DAB+ Substrate Chromogen System (Dako). Sections were viewed at 10× magnification using a microscope BZ-X710 (Keyence Corporation, Osaka, Japan). The positively stained cells were counted in three randomly selected field of view by BZ-X Analyzer (1.3.1.1., Keyence Corporation) software and the mean for each sample was calculated.
4
Histopathology and Immunohistochemistry Analysis
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Tissue sampling in this research was processed to be formalin-fixed paraffin-embedded tissue blocks. The paraffin-embedded tissue was placed in 10% buffer formalin that was cut into 4 μm sections. Next the four-mm paraffin sections were deparaffinized and stained with Hematoxyllin-Eosin to examine the histopathology of the neoplasms.
For immunohistochemistry analysis, paraffin-embedded tissues were used with the antibody α-SMA (eBioscience, Tokyo, Japan) and PCNA (Santa Cruz, Tokyo, Japan). The 4-μm paraffin sections were placed on poly-L-Lysine coated slides. After being deparaffinized, endogenous peroxidase was reduced by incubating with 3% hydrogen peroxidase in phosphate buffer saline (PBS) for 5 minutes. The secondary antibodies used were EnVision + System HRP anti rabbit (K4002, Dako, Tokyo, Japan) for α-SMA and PCNA. Diaminobenzidine was used as chromogen. Finally for counterstaining we used hematoxylin.
For immunohistochemistry analysis, paraffin-embedded tissues were used with the antibody α-SMA (eBioscience, Tokyo, Japan) and PCNA (Santa Cruz, Tokyo, Japan). The 4-μm paraffin sections were placed on poly-L-Lysine coated slides. After being deparaffinized, endogenous peroxidase was reduced by incubating with 3% hydrogen peroxidase in phosphate buffer saline (PBS) for 5 minutes. The secondary antibodies used were EnVision + System HRP anti rabbit (K4002, Dako, Tokyo, Japan) for α-SMA and PCNA. Diaminobenzidine was used as chromogen. Finally for counterstaining we used hematoxylin.
5
Histological Analysis of Cartilage Tissue
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Pellets were processed for histology by fixation in 10% buffered formalin, dehydration through graded ethanol steps, clearing in xylene, and subsequent embedding in paraffin. 5 μm sections were cut and deparaffinised before staining. Sections were stained for GAGs with 0.125% safranin-O (Merck) counterstained with 0.4% fast green (Sigma-Aldrich) and Weigert’s hematoxylin (Clin-Tech). Collagen deposition was evaluated by immunohistochemistry, with appropriate primary antibodies for type II collagen (II-II6B3; DHSB; 1:100 in PBS/BSA 5%) and type I collagen (EPR7785, BioConnect; 1:400 in PBS/BSA 5%). Samples were blocked using 0.3% H2O2, followed by antigen retrieval with pronase (1 mg/mL; Sigma-Aldrich) for 30 min at 37°C and hyaluronidase (10 mg/mL; Sigma-Aldrich) for 30 min at 37°C. Next, sections were blocked using bovine serum albumin (BSA; 5% (w/v) in PBS) for 30 min, followed by overnight incubation with the primary antibody at 4°C. After washing, type II collagen sections were incubated with a goat-anti-mouse IgG HRP-conjugated (DAKO, P0447; 1:100 in PBS/BSA 5%) and type I collagen sections with Envision + System-HRP anti-rabbit (DAKO, K4003), both for 1 h at room temperature. Next, both stainings were developed using 3,3’-diaminobenzidine (DAB, Sigma-Aldrich). Sections were counterstained with Mayer’s hematoxylin (Klinipath) and mounted in DPX mounting medium (Merck).
6
Immunohistochemical Analysis of Xenografts
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Mice xenotransplants were collected, fixed in 10% buffered formalin, embedded in paraffin, and stained with H&E (hematoxylin and eosin) according to standard protocols or processed for immunohistochemistry. Briefly, 5 μm tissue sections were dewaxed in xylene and rehydrated with distilled water. After antigen unmasking and blocking of endogenous peroxidase (3% hydrogen peroxide), the slides were incubated with rabbit anti-Ki67 (abcam, ab15580), rabbit anti-MelanA (abcam, ab51061) or rabbit anti-Vimentin (abcam, ab92547) primary antibodies. Labeling was performed using EnVision+ System-HRP anti-rabbit (Dako, K4003) and the Liquid DAB+ Substrate Chromogen System (Dako, K3468). Sections were counterstained lightly with Hematoxylin before mounting.
7
Surface Plasmon Resonance-Based HBs Antibody Detection
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HBs antigen-positive human plasma (SeraCare) and control plasma from healthy volunteers were utilized as antibody detection methods. Anti-HBs monoclonal antibody (Hs33) was fixed on a sensor chip, and a baseline spectrum was measured. Test plasma (50 µL) were subjected to an antigen-antibody reaction on a chip for 10 min, and the chip surface was washed with PBS 5 times. For signal enhancement with gold nanoparticles, 80 nm gold nanoparticle-conjugated rabbit anti-HBs antibody (50 µL; 15-fold dilution) was added, and changes in the spectrum dip were measured with a WM sensor. For signal enhancement with a peroxidase reaction, rabbit anti-HBs polyclonal antibody was added for 5 min. After washing with PBS 5 times, EnVision+System–HRP (anti-rabbit) (Dako) (no dilution) was incubated for 5 min. After further washing with PBS 5 times, AEC solution was added and changes in the spectrum dip were measured with a WM sensor.
8
Immunohistochemistry of Heparanase and ET-AR
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The paraffin-embedded tissues were used for immunohistochemistry with heparanase (AB85543, Abcam, dilution 1:200), ETAR (SC33535, Santa Cruz, dilution 1:100), and Ki-67 (CRM 325 B, Biocare Medical, dilution 1:100) antibodies. Paraffin sections, 4 μm in thickness, were placed on poly-L-lysine-coated slides. After deparaffinization, endogenous peroxidase was reduced by incubation with 3% H2O2 in phosphate buffer saline for 5 minutes. The secondary antibodies used were EnVision+System-HRP anti rabbit (K4002, Dako) for heparanase and the ETAR and Histofine SAB-PO (MULTI) (414171F, Nichirei) for Ki-67. The chromogen used was 3,3’-diaminobenzidine. Hematoxylin was utilized for counterstaining.
9
Fibromodulin Expression Analysis in Cell Aggregates
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TGFβ2- and BSA-treated cell aggregates were washed in Hanks’ balanced salt solution and then lysed in 2× sample buffer (0.5 mM Tris-HCl, 10% SDS, 2-mercaptoethanol, glycerol, pH 6.8). Cell lysates were then boiled at 95°C for 5 min and incubated on ice for another 5 min. Each protein sample (8 μl/lane) was run on 12% SDS-PAGE and transferred electrophoretically to an Immobilon-P transfer membrane (Millipore, Bedford, MA, USA). The blots were incubated with 5% skim milk in T-TBS (50 mM Tris-HCl, 100 mM NaCl, 0.1% Tween 20, pH 7.4) for 1 hr and probed overnight with rabbit polyclonal anti-fibromodulin antibody (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in Can Get Signal Solution (Toyobo, Osaka, Japan), followed by incubation for 1 hr with horseradish peroxidase (HRP)-labeled secondary antibodies (Envision+System-HRP, anti-rabbit, Dako). Anti-β-actin antibody (0.1 μg/ml; BioVision. Mountain View, CA, USA) was used as a control.
Immunoreactive bands were visualized by ECL plus Western Blotting Detection Reagents (GE Healthcare,Mississauga , ON, Canada) with an LAS-3000 imaging system (Fujifilm, Tokyo, Japan). The acquired images were analyzed by densitometry using ImageJ software (NIH, Bethesda, MD, USA). The data were normalized with β-actin.
Immunoreactive bands were visualized by ECL plus Western Blotting Detection Reagents (GE Healthcare,
10
Immunohistochemical Analysis of Tumor Proteins
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Tumor blocks collected before therapy were available for a limited number of patients only. Two μm serial tissue sections were cut from formalin-fixed, paraffin-embedded tumor tissues, dewaxed, and rehydrated according to standard procedure (preheating at 60°C; deparaffinization in Neo-Clear, Merck KGaA; rehydration in graded series of ethanol and distilled water) and stained with hematoxylin and eosin for determination of areas of tumor necrosis. For IHC analysis, the primary antibodies anti-GPx3 (goat IgG polyclonal, dilution 5 μg/mL; R&D Systems; Catalog No. AF4199) and ACTR3 (rabbit monoclonal, clone JB33–44, dilution 1:200; Novus Biologicals; Catalog No. JB33–44) were applied overnight at 4°C, followed by incubation with secondary antibodies (for GPx3: Peroxidase AffiniPure Donkey Anti-Goat; Jackson ImmunoResearch Europe Ltd.; for ACTR3: Dako EnVision+System-HRP Anti-Rabbit; Carpinteria). DAB substrate kit (DAB Substrate Kit; Cell Signaling Technology) was used as chromogen. Sections were counterstained with hematoxylin, dehydrated, and mounted using Neo-Mount (Merck KGaA).
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