Immunohistochemical staining of TMAs was performed with BPGAP1 (in-house production) and VAV1 antibodies (Acris-OriGene; Catalogue: AP06359PU-N) using the Bond Max automated immunohistochemistry vision Biosystem (Leica Microsystems GmbH, Germany) based on the manufacturer’s protocol. TMAs were first deparaffinized, and antigen retrieval was conducted by boiling the slides with Bond Epitope Retrieval Solution 2 (citrate-based pH 9.0 solution) at 98°C for 20 min. This was followed by peroxidase blocking using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH, Germany). TMAs were washed once before incubation with the BPGAP1 (1:50) or VAV1 (1:100) antibodies for 30 min. Finally, polymer was added, and the staining color was developed using DAB-Chromogen.
Bond polymer refine detection kit dc9800
Manufactured by Leica
Sourced in Germany
The Bond Polymer Refine Detection Kit DC9800 is a laboratory equipment product designed for use in immunohistochemistry (IHC) applications. It provides a polymer-based detection system that enhances the visualization of target antigens in tissue samples.
Automatically generated - may contain errors
5 protocols using bond polymer refine detection kit dc9800
1
Immunohistochemical Staining of TMAs
2
Immunohistochemistry for p16 and DCC
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Immunohistochemistry was performed using 4-μm-thick FFPE tissue sections from the derivation cohort and primary monoclonal antibodies for p16 (E6H4, prediluted; CIN Histology Kit, Roche, Heidelberg, Germany) as a surrogate marker for human papilloma viruses, and DCC (G97-449, dilution 100; BD Pharmingen, Franklin Lakes, NJ, USA). All stages of immunohistochemical staining were performed automatically using a BOND-MAX Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany). Tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 2 (EDTA-buffer pH8.8) at 98 °C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed and then incubated with the primary antibody for 30 min. The immunohistochemical staining was observed by optical microscope and the p16 protein expression was considered positive (protein overexpression) when ≥70% of the tumor cells showed strong diffuse nuclear and cytoplasmic staining.
3
Immunohistochemical Analysis of Rab35 Protein
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Immunohistochemical staining was performed as follows: 3-μm-thick sections were prepared from formalin-fixed paraffin-embedded TMA tissue blocks and dried in a 37 °C oven overnight. The sections were placed in a Bond Max Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 2 (pH9) at 100 °C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed and then incubated for 30 min with the primary antibody diluted (1:50 anti-Rab35, Abcam ab 152138) in Bond Primary Antibody Diluent (AR9352). Subsequently, tissues were incubated with post primary and polymer for a total of 16 min and developed with DAB-Chromogen for 10 min and counterstained with haematoxilin for 5 min.
Human samples were collected with inform consent between 1995 and 2010, unfortunately, without recording their clinico-pathological features, thus impeding the analysis of RAB35 expression levels with relevant tumour properties.
Human samples were collected with inform consent between 1995 and 2010, unfortunately, without recording their clinico-pathological features, thus impeding the analysis of RAB35 expression levels with relevant tumour properties.
4
Immunostaining of Tumor Microenvironment
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This article is protected by copyright. All rights reserved Three double immunostains were performed, namely the pankeratin cocktail AE1/AE3 to highlight tumour cells and CD8, CD3 or CD45RO, respectively. Double immunostains ngTMA blocks containing cores from tumour centre, front and microenvironment using the BOND-RX Automated System (Leica Microsystems GmbH, Newcastle, United Kingdom). ngTMA blocks were cut at 4 µm, deparaffinised and pre-treated with the Epitope Retrieval Solution 2 (Citratebuffer pH 8.8) at 100 C° for 20min. After wash steps, peroxidase blocking was carried out for 4 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed, then incubated with primary antibody against AE1/AE3 with the dilution 1:200 (DakoCytomation, Glostrup, Denmark) for 30min. Subsequently, tissues were incubated with polymer for 15 min and then with DAB-Chromogen for 10min (Bond Polymer AP Red Detection Kit DS9305, Leica Microsystems GmbH). After washing, incubation was carried out with anti-CD8 (DakoCytomation, Glostrup, Denmark), anti-CD3 (Abcam, Cambridge) and anti-CD45RO (Abcam, Cambridge) with the dilutions CD8 1 :100, CD3 1 :400, CD45RO 1 :5000 for 30 min (reduced to 15 min in optimizing the CD45RO protocol) followed by application of AECsubstrate for 10 min and counterstaining with haematoxylin for 5 min.
5
Immunohistochemical Staining of COVID-19 Lung Tissue
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Immunohistochemical staining was performed as follows: 3-µm-thick sections were prepared from formalin-fixed paraffin-embedded autoptic tissue lung blocks from a COVID-19 patient and were dried in a 60°C for 20'. The sections were placed in a BOND-III Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 1
(Citrate-buffer pH 5.9-6.1) at 100°C for 10 min. After washing steps, peroxidase blocking was carried out for 5 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH).
Tissues were again washed and then incubated with the primary antibody (affinity purified rabbit IgG anti-human PTX3) 39 for 15 min. Subsequently, tissues were incubated with polymer for 8 min and developed with DAB-Chromogen for 10 min.
(Citrate-buffer pH 5.9-6.1) at 100°C for 10 min. After washing steps, peroxidase blocking was carried out for 5 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH).
Tissues were again washed and then incubated with the primary antibody (affinity purified rabbit IgG anti-human PTX3) 39 for 15 min. Subsequently, tissues were incubated with polymer for 8 min and developed with DAB-Chromogen for 10 min.
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