For immunohistochemistry, the slides were immersed in an antigen retrieval solution consisting of EDTA/Tris, pH 9.0, at 125 °C in an electric pressure cooker managed by a REPTIDE microprocessor (Celerus). Nonspecific binding of the antibodies was blocked by incubation of the slide with hydrogen peroxide. Next, the slides were incubated with the primary ADPH antibody (clone AA5-27, LifeSpan) diluted 1/100. To amplify the antigen–antibody binding, two other antibodies were applied to the slides, post-primary (Novolink polymer Novocastra) and the polymer (Novolink polymer Novocastra). The reaction was developed with diaminobenzidine (DAB) as a chromogen (Liquid DAB + Substrate Chromogen System Kit, DakoCytomation, Carpinteria, CA, USA).
Novolink polymer
Manufactured by Leica
Sourced in United Kingdom, Germany, Denmark, United States
The Novolink Polymer is a ready-to-use, one-step polymer detection system designed for use with Leica's range of immunohistochemistry (IHC) and in situ hybridization (ISH) instruments. It provides a reliable and efficient method for the visualization of targeted antigens or nucleic acid sequences in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ferangi blue, by Biocare Medical (5 mentions)
Mach 4 mr ap, by Biocare Medical (4 mentions)
Protein block, by Leica (3 mentions)
Diaminobenzidine dab, by Agilent Technologies (2 mentions)
B220 antibody clone ra3 6b2, by BD (2 mentions)
Novocastra post primary, by Leica (2 mentions)
3 3 diaminobenzidine chromogen dab, by Leica (2 mentions)
24 pax 5, by BD (2 mentions)
Novolink polymer detection kit, by Leica (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Ultravision quanto detection system hrp polymer, by Thermo Fisher Scientific (1 mentions)
Re7101, by Leica (1 mentions)
8f1 clone, by Thermo Fisher Scientific (1 mentions)
G59 12 clone, by BD (1 mentions)
Diaminobenzedine substrate, by Leica (1 mentions)
Liquid dab substrate chromogen system kit, by Agilent Technologies (1 mentions)
Ab150160, by Abcam (1 mentions)
Ab64226, by Abcam (1 mentions)
Ds ri2 camera, by Nikon (1 mentions)
Anti pdgfrα antibody, by Santa Cruz Biotechnology (1 mentions)
34 protocols using novolink polymer
1
Immunohistochemical Staining Protocol
2
Comprehensive Immunohistochemical Profiling of Tumor-associated Immune Cells
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Immunohistochemistry (IHC) was performed on a set of tissues samples obtained from the archive of the Department of Pathology (Policlinico San Martino, Genova). For each patient, tissues included a representative whole‐tissue formalin‐fixed, paraffin‐embedded (FFPE) block of primary carcinoma. Immunostaining was performed using 4 μm‐thick tissue sections and a set of primary antibodies (Supplementary table 1 ).
The reaction was revealed using Novolink Polymer (Leica Microsystems) followed by diaminobenzidine (DAB, Dako, Glostrup, Denmark). Finally, the slides were counterstained with Meyer's Haematoxylin.
For double staining, after completing the first immune reaction, the second was visualised using Mach 4 MR‐AP (Biocare Medical), followed by Ferangi Blue (Biocare Medical). For triple IHC, the third immune reaction was revealed using Novolink Polymer (Leica Microsystems), and developed in 3‐amino‐9‐ethylcarbazole chromogen (AEC), counterstained with haematoxylin and cover‐slipped using gelatin. Single staining for CD3, CD8, CD20, CD66b, CD163 and CD38 was applied to identify T cells, B cells, neutrophils, macrophages and plasma cells, respectively. To characterise the phenotype and the localisation of tumor‐associated B cells, we performed double and triple immunostaining for a set of B‐cell markers including CD20, BCL6, CD27, PAX5, MUM1, CD38, FOXP3, PD‐L1 and pSMAD2.
The reaction was revealed using Novolink Polymer (Leica Microsystems) followed by diaminobenzidine (DAB, Dako, Glostrup, Denmark). Finally, the slides were counterstained with Meyer's Haematoxylin.
For double staining, after completing the first immune reaction, the second was visualised using Mach 4 MR‐AP (Biocare Medical), followed by Ferangi Blue (Biocare Medical). For triple IHC, the third immune reaction was revealed using Novolink Polymer (Leica Microsystems), and developed in 3‐amino‐9‐ethylcarbazole chromogen (AEC), counterstained with haematoxylin and cover‐slipped using gelatin. Single staining for CD3, CD8, CD20, CD66b, CD163 and CD38 was applied to identify T cells, B cells, neutrophils, macrophages and plasma cells, respectively. To characterise the phenotype and the localisation of tumor‐associated B cells, we performed double and triple immunostaining for a set of B‐cell markers including CD20, BCL6, CD27, PAX5, MUM1, CD38, FOXP3, PD‐L1 and pSMAD2.
3
Immunostaining of Pancreatic Tumor Tissue
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Tissue microarrays were produced from paraffin-embedded tissue blocks from 41 PDAC surgical specimens. All cases were immunostained with a sensitive non-biotin detection system (NovoLink polymer, Novocastra), with diaminobenzidine development. Heat induced antigen retrieval was performed using citrate buffer 0.01 M, pH 6.0 or Tris-EDTA pH 9.0 in a water bath for 30′. Immunostaining was performed using the anti-ASC Ab (Enzo Life Science) and Ultravision Quanto Detection System HRP polymer (Thermo Scientific). Samples were considered positive if the percentage of positive cells was superior to 10%.
4
Immunohistochemistry Protocols for Protein Expression
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For immunohistochemistry, TMA was constructed, as described by Pires et al. (19 (link)). All samples were fixed in 10% formalin and embedded in paraffin. Serial TMA sections were cut, mounted on glass slides and dried at 56°C before dewaxing in xylene and rehydration in alcohol. All sections were subjected to heat-induced epitope retrieval in citrate buffer followed by inhibition of endogenous peroxidase (peroxidase block, RE7101, Novocastra, UK). Incubation of primary antibodies against EGFR (D38B1, 1:25, Cell Signaling Technology), ERCC1 (1:100; 8F1 clone, Thermo Fisher Scientific), or p53 (1:100; G59–12 clone, BD Pharmingen) was performed for 1 h. Slides were incubated with NovoLink™ polymer (RE7112, Novocastra), further developed with diaminobenzidine chromogen (RE7105, Novocastra), and finally stained with Mayer hematoxylin, dehydrated and mounted with Canadian balsam. EGFR, ERCC1 and p53 expressions were evaluated semi-quantitatively as positive cells after counting 300–500 tumor cells, being scored as negative (no staining), 1+ (<25% of positive cells), 2+ (25–75% of positive cells) or 3+ (>75% cells staining positively). Negative controls were obtained by omitting the primary antibody.
5
Immunostaining of Heart Sections
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Immunostaining of the heart sections for the detection of pJAK2 and pSTAT3 was performed using streptavidin-biotinylated horseradish peroxidase (Novalink Max Polymer detection system; Novocastra Laboratories, Newcastle, UK). In brief, sections were incubated in 3% hydrogen peroxide in distilled water for 5 minutes to block endogenous peroxidase activity and then washed in Tris-buffered saline (pH 7.6) for 10 minutes. Nonspecific binding of antibodies was blocked by incubation with protein block (Novocastra) for 5 minutes. Sections were incubated with rabbit polyclonal anti-pJAK2 and goat polyclonal anti-pSTAT3 primary antibodies (Santa Cruz Biotechnology Inc.) diluted 1:100 at room temperature for 1 hour. The sections were washed in Tris-buffered saline three times and incubated with biotinylated IgG (Novocastra) for 30 minutes. After washing in Tris-buffered saline and incubation with Novolink polymer (Novocastra) for 30 minutes, peroxidase was detected with diaminobenzedine substrate (Novocastra). Sections were then washed in distilled water, stained with Mayer’s hematoxylin, and mounted in DPX. The same procedure, with the omission of incubation in primary antibodies, was followed for negative control sections.
6
Immunostaining of Cerebellar Sections
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Immunostaining was done by dehydration and de-waxing of paraffin blocks then cerebellar sections of 5 µm thick were cut and buffered with 10-mM sodium citrate for antigen retrieval. 0.9% hydrogen peroxide in absolute methanol was used for blocking activity of internal peroxidase. Not related antibodies were blocked by addition of protein for 1 h (ab64226, Abcam, Cambridge, UK). Incubation of sections was done with mouse monoclonal GFAP antibodies (Abcam, Cambridge, UK) 1:20 dilution and rabbit monoclonal caspase-3 antibodies (Abcam, Cambridge, UK), 1:200 dilutions for 1 h. Sections were rinsed in phosphate buffered saline (PBS) then heated with Rat IgG antibody (ab150160, Abcam, Cambridge, UK), followed by washing in PBS and incubation with Novolink polymer (Novo-castra). Deletion of the primary antibody in automated staining design and tonsillar human tissue were done for the 2 negative controls for GFAP and caspase-3.
7
Immunohistochemical Analysis of Placental Tissue
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Formalin-fixed paraffin-embedded tissue blocks used for this study were retrieved from the tissue bank of the Department of Pathology (ASST-Spedali Civili di Brescia, Brescia, Italy). Tissues used for the analysis included normal placental tissue at the first (five cases) and third (ten cases) trimester. Four-micron-thick tissue sections were used for immunohistochemical staining. Primary antibodies included anti-CD14 (1:50, mouse, clone 7, Leica); anti-CD163 (1:50, mouse, clone 10D6, Neomarkers); and anti-HLA-DP,DQ,DR(1:500, mouse, clone CR3/43, DAKO). The reaction was revealed using Novolink Polymer (Leica Microsystems) followed by DAB. Microphtalmia-associated transcription factor (MITF) (1:50, mouse, clone D5, DAKO) was visualized using Mach 4 MR-AP (Biocare Medical), followed by Ferangi Blue (Biocare Medical) as chromogen.
8
Immunohistochemistry Protocol for FFPE Tissues
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Briefly, 2-μm-thick paraffin sections were obtained from FFPE. Sections were de-waxed, re-hydrated and endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 20 min. Antigen retrieval (when necessary) was performed using a microwave or a thermostatic bath in either 1.0 mM EDTA buffer (pH 8.0) or 1 mM Citrate buffer (pH 6.0). Sections were then washed in TBS (pH 7.4) and incubated for one hour or overnight in the specific primary antibody diluted in TBS 1% bovine serum albumin. The primary antibodies used are listed in Table S1 . The reaction was revealed by using Novolink Polymer (Leica Microsystems GmbH, Wetzlar, Germany) or Dako EnVision+Dual Link System Peroxidase (DakoCytomation, Glostrup, Denmark) followed by DAB and slides counterstained with Hematoxylin. Images were acquired with a Nikon DS-Ri2 camera (4908 × 3264 full-pixel) mounted on a Nikon Eclipse 50i microscope equipped with Nikon Plan lenses (10×/0.25; 20×/0.40; 40×/0.65; 100×/1.25) using NIS-Elements 4.3 imaging software (Nikon Corporation, Tokyo, Japan).
9
Immunohistochemical Staining of PDGFRα in Prostate Tissue
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Sections were deparaffinized in toluene, rehydrated with ethanol, and immersed in citrate antigenic retrieval buffer during 20 min in 95 °C water-bath and then cooled at room temperature for 20 min. Endogenous peroxidase activity was subsequently blocked with 3% hydrogen peroxide in methanol followed by incubation with protein block for 30 min. The sections were incubated with the primary antibody: anti-PDGFRα antibody (1:100; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. After phosphate-buffered saline (PBS) washing, tissue sections were incubated with biotinylated secondary antibody during 30 min, followed by incubation with novolink polymer (Leica Microsystems, Newcastle Ltd.) for 30 min. The antibody complex was visualized by the chromogens 3-amino-9-ethylcarbazole (AEC) and sections were counterstained with Mayer’s hematoxylin. Prostate tissue from department of Human and Experimental pathology of Institut Pasteur de Tunis was used as a positive control for primary antibody. For negative controls, the anti-PDGFRα antibody was replaced by PBS.
10
Immunohistochemical Evaluation of Claudin-7
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Four-micron sections from formalin-fixed, paraffin-embedded (FFPE) tissues were stained for claudin-7 after antigen retrival by microwave treatment in EDTA buffer-pH 8.0, applying anti-Claudin-7 (mouse, clone 5D10F3, 1:80, Thermo Scientific) for 60 min. The reaction was revealed using Novolink Polymer (Leica Microsystem) or Envision System-HRP Labeled Polymer anti-Mouse (Dako) followed by DAB.
Immunoreactivity was evaluated by four independent observers (CR, LA, MB, EB); staining was graded for intensity (1–3+) and percentage of stained cells, and a single H score was obtained by multiplying the intensity and the percentage staining. Positivity was defined by at least 1+ intensity in 10% or more of the cells (H-score = 10).
Immunoreactivity was evaluated by four independent observers (CR, LA, MB, EB); staining was graded for intensity (1–3+) and percentage of stained cells, and a single H score was obtained by multiplying the intensity and the percentage staining. Positivity was defined by at least 1+ intensity in 10% or more of the cells (H-score = 10).
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