The entire lung was removed and fixed in 3.7% neutral buffered formalin (pH 7.4; Sigma-Aldrich) for 2 days. Specimens were embedded in paraffin and sliced at a thickness of 4 μm. H&E (hematoxylin and eosin) staining was performed according to the manufacturers’ protocols (ScyTek Laboratoriea, Logan, UT, USA). Lung sections were deparaffinized for immunostaining. After antigen retrieval and non-specific binding blocking, the samples were incubated with anti-iNOS (anti-inducible nitric oxide synthase; GeneTex) and anti-arginase-1 antibodies (GeneTex). Immunoreactivity was visualized using the diaminobenzidine immunoperoxidase methodology with the Novolink™ Polymer Detection Systems kit (Leica, Wetzlar, Germany).
Novolink polymer detection systems kit
Manufactured by Leica
Sourced in Germany, United Kingdom, Australia
The NovoLink Polymer Detection Systems kit is a laboratory equipment product designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. The kit provides a polymer-based detection system that enhances the visualization of target antigens or nucleic acid sequences in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Bond er1 solution, by Leica (2 mentions)
Anti inos, by GeneTex (1 mentions)
Cv ultra mounting medium, by Leica (1 mentions)
Axioscan z1, by Definiens (1 mentions)
Grandmaster, by 3DHISTECH (1 mentions)
Scanscope cs, by Leica (1 mentions)
Mounting medium, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Ab133616, by Abcam (1 mentions)
Ab20034, by Abcam (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Ab125286, by Abcam (1 mentions)
16 protocols using novolink polymer detection systems kit
1
Immunostaining for iNOS and Arginase-1 in Lung Tissue
2
Immunohistochemical Analysis of Osteosarcoma
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Normal human bone tissue and OS specimens were obtained from China Medical University Hospital (Institutional Review Board protocol code CMUH 104-REC3-110). Tissue microarray slides for OS cases of different stages were purchased from US Biomax, Inc (Rockville, MD, USA). Immunohistochemistry was performed using the Novolink Polymer Detection Systems kit (Code: RE7280-K, Leica, Biosystems, UK) following the manufacturer's protocols, as previously described 33 . Immunoreactive signals were quanti ed using ImageJ software.
3
Immunohistochemistry Protocol for Tissue Analysis
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Immunohistochemistry analysis was performed at the neuropathology laboratory, Royal Brisbane and Women’s Hospital, QLD, Australia. Immunohistochemistry used a Leica Novolink Polymer Detection Systems Kit (Leica Microsystems Pty Ltd., North Ryde, NSW, Australia) as per manufacturer’s instructions, www.leica-microsystems.com . Sections had paraffin removed through a series of xylene immersions and re-hydrations. Antigen retrieval was carried out using Leica BOND ER1 solution. Endogenous peroxidize was neutralized. Sections were incubated with a protein block. The primary antiserum made up in Leica BOND Antibody Diluent was applied to the sections.
4
Immunohistochemistry for Tissue Analysis
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Immunohistochemistry analysis was performed at the neuropathology laboratory, Royal Brisbane and Women’s Hospital, QLD, Australia, using a Leica Novolink Polymer Detection Systems Kit (Leica Microsystems Pty Ltd., North Ryde, 2113 Australia) as per the manufacturer’s instructions. Paraffin was removed from the sections through a series of xylene immersions and re-hydrations. Antigen retrieval was carried out using Leica BOND ER1 solution. Endogenous peroxidase was neutralized. Sections were incubated with a protein block. The primary antiserum made up in Leica BOND Antibody Diluent was applied to the sections.
5
Immunohistochemistry Analysis in Neuropathology
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Immunohistochemistry analysis was performed at the neuro-pathology laboratory, Royal Brisbane and Women’s’ Hospital, QLD, Australia. Immunohistochemistry used a Leica Novolink Polymer Detection Systems Kit (Leica Microsystems Pty Ltd., North Ryde, 2113 Australia) as per manufacturer’s instructions, www.leica-microsystems.com . Sections had paraffin removed through a series of xylene immersions and re-hydrations. Antigen retrieval was carried out using Leica BOND ER1 solution. Sections were incubated with a protein block. The primary antiserum made up in Leica BOND Antibody Diluent was applied to the sections.
6
Immunohistochemical Analysis of E-Cadherin
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To evaluate the expression of E-cadherin, we performed immunohistochemical staining on formalin-fixed and paraffin-embedded (FFPE) samples of gastric tumor tissues. Immunostaining was performed with a primary mouse monoclonal against E-cadherin (NCL-L-E-Cad, clone 36B5, Novocastra TM, Biopole), recognizing the external Nt domain, according to the manufacturer’s instructions using a Novolink Polymer Detection Systems kit (Leica Biosystems, United States/Biopole, Tunisia).
7
Immunohistochemical Staining and Cartilage Analysis
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IHC staining was performed as described in our previous studies [47 (link),48 (link)]. In brief, the sections were incubated with primary antibodies specific for mouse IL-1β, BMP-2, and MMP-13. The immunodetection assay was performed using a NovoLink Polymer Detection Systems kit (Leica Biosystems, Wetzlar, Germany), following the manufacturer’s procedures. We also used Safranin O/Fast Green staining to evaluate the cartilage structure lesion of specimens from ACLT rat knees. Slides were observed under a light microscope and digitized with a Pannoramic scanner digital microscope (3DHISTECH, Budapest, Hungary). Images were obtained from digitized slides with the CaseViewer software (v 2.3, 3DHISTECH, Budapest, Hungary).
8
Cartilage Degeneration Analysis in Rats
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Paraffin-embedded sections were prepared from sacrificed rats. The sections were subjected to Safranin O/fast green staining to evaluate cartilage degradation, or incubated with a specific antibody against IL-6, as per our previous protocol.22 (link) IL-6 antibody in the sections was detected by the NovoLink Polymer Detection Systems kit (Leica Biosystems, Wetzlar, Germany), according to the manufacturer’s protocol.
For Safranin O/fast green staining, briefly, the sections were stained with Safranin O/fast green, hematoxylin, and eosin to investigate the histopathological changes of all experiment groups under a light microscope. The cartilage destruction was evaluated according to the OARSI score system established by the International Association for Osteoarthritis Research.35 (link) The OARSI score system includes 6 grades (Grade 0 = no cartilage degeneration; Grade 1 = Minimal degeneration, 5–10% of the total projected cartilage area affected by matrix or chondrocyte loss; Grade 2 = Mild degeneration, 11–25% affected; Grade 3 = Moderate degeneration, 26–50% affected; Grade 4 = Marked degeneration, 51–75% affected; Grade 5 = Severe degeneration, greater than 75% affected). The scoring was evaluated blindly by two individuals and the scores were averaged to minimize observer bias.
For Safranin O/fast green staining, briefly, the sections were stained with Safranin O/fast green, hematoxylin, and eosin to investigate the histopathological changes of all experiment groups under a light microscope. The cartilage destruction was evaluated according to the OARSI score system established by the International Association for Osteoarthritis Research.35 (link) The OARSI score system includes 6 grades (Grade 0 = no cartilage degeneration; Grade 1 = Minimal degeneration, 5–10% of the total projected cartilage area affected by matrix or chondrocyte loss; Grade 2 = Mild degeneration, 11–25% affected; Grade 3 = Moderate degeneration, 26–50% affected; Grade 4 = Marked degeneration, 51–75% affected; Grade 5 = Severe degeneration, greater than 75% affected). The scoring was evaluated blindly by two individuals and the scores were averaged to minimize observer bias.
9
Immunohistochemical Analysis of Mouse Brain
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Paraformaldehyde-fixed mouse brains were paraffin embedded and sectioned (4 μm) for IHC analysis and stained with hematoxylin and eosin. For IHC, sodium citrate (pH 6.0) heat-mediated antigen retrieval was performed, and staining was carried out using an antibody directed against human nuclear antigen (Millipore, #4383, 1:100). Pressure antigen retrieval was performed and staining was carried out using antibodies directed against Ki67, (DAKO, #7240, 1:100) and CD31 (Abcam, #28364, 1:50). All primary antibodies were diluted into 1% Tris buffer solution with 0.05% Tween-20, except Ki67, which was diluted into Dako antibody diluent. Antibodies were incubated for 1 hour at room temperature. A Novocastra Novolink Polymer Detection Systems Kit (Leica Biosystems RE-7150) was used for the staining. Slides were then mounted using a Leica CV Ultra mounting medium, and slides were imaged using the high-throughput scanning microscope AxioScan Z1 and quantified using Definiens software.
10
Lung Tissue Immunohistochemistry Protocol
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Following the sacrifice of mice, their lung tissues were collected and preserved in formalin solution (10%). After fixation, dehydration was achieved by employing a mixture of xylene and ethanol. The tissue samples underwent embedding into paraffin blocks, which were then sliced to obtain sections with a thickness measuring 5 μm each. Subsequently, these sections were subjected to incubation using primary antibodies diluted at a ratio of 1:100 for 1 h under room temperature conditions. Signal detection within these sections was achieved via employment of the NovoLink Polymer Detection Systems kit (Leica Biosystems, Wetzlar, Germany) according to instructions provided by the manufacturer. The staining results were photographed across three random fields for each slide analysed.
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