FFPE tumor sections were dewaxed in xylene and ethanol and submerged into ER1 buffer (pH 6.0) for 20 min at 100°C in a Bond-RX Multiplex IHC Stainer (Leica Biosystem, Melbourne, Australia) to retrieve the antigens, followed by incubation in endogenous peroxidase for 10 min. Anti-HLA-DPB1 antibody (Abcam, Cambridge, UK) was diluted at 1:1,000 and incubated with Bond-RX autoimmunostainer for 15 min. For the HLA-DRB1 (IHC) test, we diluted the anti-HLA-DRB1 antibody (GeneTex, Irvine, CA, USA) to 1:2,000 and incubated it with Bond-RX autoimmunostainer for 15 min. For the HLA-DQA1 test, we diluted the anti-HLA-DQA1 antibody (Abcam) to 1:200 and incubated it with Bond-RX autoimmunostainer for 15 min. Subsequently, the immunostained slides were evaluated by an experienced pathologist. IHC scores were assessed by both staining intensity and the percentage of positive tumor cells. The staining intensity was graded from 0 to 3 (0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining) by relative degree within each antibody. The percentage of positive staining cells was recorded from 0 to 100%. In addition, we evaluated the quality of the IHC test depending on the staining of immune cells (internal control).
Bond rx multiplex ihc stainer
Manufactured by Leica
Sourced in Germany
The Bond-RX Multiplex IHC Stainer is a fully automated immunohistochemistry (IHC) staining system designed for multiplex staining. It performs automated staining, dewaxing, and epitope retrieval for a wide range of IHC markers.
Automatically generated - may contain errors
Lab products found in correlation
Fluoromount g, by Southern Biotech (2 mentions)
Cd8 sk1, by BioLegend (1 mentions)
Superfrost microscope slide, by Thermo Fisher Scientific (1 mentions)
Cd4 okt4, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Akoya Biosciences (1 mentions)
Cd20 2h7, by BioLegend (1 mentions)
Opal multiplex ihc detection kit, by Akoya Biosciences (1 mentions)
Cd69 fn50, by BioLegend (1 mentions)
Kma 0100 00a, by CellPath (1 mentions)
Vectra polaris, by Akoya Biosciences (1 mentions)
Halo software, by Indica Labs (1 mentions)
Cd206, by Agilent Technologies (1 mentions)
Cd163, by Abcam (1 mentions)
Evos xl core, by Thermo Fisher Scientific (1 mentions)
6 protocols using bond rx multiplex ihc stainer
1
IHC Evaluation of HLA Antigen Expression
2
Multiplex Immunofluorescence Analysis of Tissue-Resident Immune Cells in Lung
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Human and NHP lung tissues were placed in optimal cutting temperature compound (OCT, KMA-0100-00A, CellPath, UK) and snap frozen in liquid nitrogen. 10 µm sections were cut on to Super Frost microscope slides (12312148, Thermo Fisher) using a cryostat. Sections were air dried overnight at room temperature and then fixed in -20°C acetone for 10 minutes. Sections were air dried for <20 minutes prior to x2 washes with PBS. Multiplex immunofluorescence was performed on lung sections using a fully automated Bond-Rx Multiplex IHC Stainer (Leica Biosystems) and OPAL Multiplex IHC Detection Kit and counterstained with DAPI (Akoya Biosciences) according to a previously published protocol (27 (link)). TRM were stained with purified, cross-reactive CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD69 (FN50, Biolegend) and CD103 (2G5.1, Bio-Rad, UK) antibodies. BRM were stained with purified, cross-reactive CD20 (2H7, Biolegend), CD27 (M-T271, Biolegend) and CD69 (FN50, Biolegend) antibodies. Human lung (n=5) and NHP lung (n=3) sections were stained simultaneously using the same protocol and antibody concentrations. For full details, see online supplement.
3
PD-L1 Expression Evaluation by IHC
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PD-L1 immunohistochemistry (IHC) was performed using the BOND-RX Multiplex IHC Stainer (Leica Biosystems, Nussloch, Germany; Data Supplement, Methods). Assessment of PD-L1 expression was performed by two surgical pathologists blinded to the genomic results of the respective samples (J.S.R.-F. and F.P.).
4
Multiplex Immunohistochemistry Analysis of Tumor-Infiltrating Immune Cells
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Tumor-infiltrating leukocytes (including T cells, B cells, tumor associated macrophages, and NK cells) and tertiary lymphoid structures were detected using mIHC staining in the two groups of samples. Staining and imaging were performed using an automated staining instrument (BOND-RX Multiplex IHC Stainer, Leica, Wetzlar, Germany) and a quantitative pathology analysis system (Vectra Polaris, Akoya Biosciences, Marlborough, MA, USA). Antibody kits used included CD163: ab182422; CD56: ab75813; CD68: ab213363; CD8: ab178089; PD-L1: E1L3N (13684S); CK: ab7753; and S100: ab52642. All kits were from Bainuo Panorama Biotechnology (Beijing, China). HALO software (Indica Labs, Albuquerque, NM, USA) was used for tumor tissue and cell recognition, and for providing the density and percentages of cells positive for the different marker molecules in the intratumoural region (IT) and tumour rim (TR). Additionally, HALO was used to analyse the relationship between abnormal HER2 amplification and immune cell infiltration and diseases prognosis when compared with the control group.
5
Multiplex IHC Staining for Immune Cell Profiling
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Tissue sections were deparaffinized by 1 h incubation at 60°C. Tissue sections (5 µm) were stained with Opal 7-Color Automation immunohistochemistry Kits (Akoya Bioscience) in the BOND-RX Multiplex IHC Stainer (Leica). The first step of the automated IHC included a fixation step with 4% Formalin (Roth, P087.5) in PBS. Each section was put through 6 sequential rounds of staining, which included blocking in 5% BSA, followed by incubation with primary antibodies of the following panel (NT, Santa Cruz, sc-32757; NOS2, Santa Cruz, sc-7271; CD163, Abcam, ab182422; CD206, Cell signaling, 91992S; CD68, DAKO, M0876), corresponding secondary HRP-conjugated antibodies and Opal fluorophores as described. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) contained in the Opal 7-Color Automation IHC Kits, and slides were mounted with Fluoromount-G (SouthernBiotech).
6
Quantification of Hepatic Steatosis and Fibrosis
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For histochemistry, mice livers were perfused to remove blood and were fixed with a zinc fixative (100 mM Tris (pH 7.4) with 3.2 mM calcium acetate, 27.3 mM zinc acetate and 36.7 mM zinc chloride) for 6–8 h at 4°C. Afterwards, paraffin slides were created and underwent H&E staining. Images were created on an EVOS XL Core (Thermo Fisher) at 40× magnification. The software ImageJ was used to apply scale bars. Forty fields of a single H&E stained liver slice per time point were assessed by an expert liver pathologist to quantify hepatic steatosis, which was classified in grade 0–2 (i.e., 0%, ≤ 50% or >50% of hepatocytes are steatotic). Histological Sirius red staining and analysis for hepatic fibrosis quantification was performed as described previously (22 (link)). For immunofluorescence analysis of neutrophil infiltrates in the liver, sections were stained with anti-Ly6G antibody (Biolegend, 1A8), and Opal 650 reagent pack in the BOND-RX Multiplex IHC Stainer (Leica). Nuclei were counterstained with DAPI and slides were mounted with Fluoromount-G (SouthernBiotech). Slides were imaged at 20× using Vectra3 imaging system and analyzed using inForm 2.4.9 software (both from Akoya Biosciences).
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