Tissues were cut into 5-μm sections, and multiple sections were placed on each slide for analysis. For immunohistochemistry, slides were deparaffinized, and heat-induced antigen retrieval was performed on the Bond Max automated IHC stainer (Leica Biosystems, Buffalo Grove, IL) using their Epitope Retrieval 2 solution for 5 to 20 min. Slides were incubated with a rabbit polyclonal anti-GBS antibody (ab78846; Abcam), rabbit polyclonal anti-histone H3 antibody (ab8580; Abcam), or a mouse monoclonal anti-CD163 antibody (MRQ-26; Cell Marque, Rocklin, CA) for 1 h. The Bond Polymer Refine detection system (Leica Biosystems) was used for visualization. Slides were dehydrated and cleared, and coverslips were added before light microscopy analysis was performed.
Mrq 26
Manufactured by Cell Marque
Sourced in United States
The MRQ-26 is a laboratory equipment product from Cell Marque. It is designed for use in various research and diagnostic applications. The core function of the MRQ-26 is to provide a controlled environment for sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab78846, by Abcam (1 mentions)
Ab8580, by Abcam (1 mentions)
Bond polymer refine detection system, by Leica Biosystems (1 mentions)
Benchmark ultra slide staining system, by Roche (1 mentions)
Pg m1, by Agilent Technologies (1 mentions)
Blocking buffer, by Agilent Technologies (1 mentions)
Ab64693, by Santa Cruz Biotechnology (1 mentions)
Sp142, by Leica Biosystems (1 mentions)
Nel811001kt, by PerkinElmer (1 mentions)
Nef812001ea, by PerkinElmer (1 mentions)
Nef822001ea, by PerkinElmer (1 mentions)
Clone e1l3n, by Cell Signaling Technology (1 mentions)
Software bvba version 12, by MedCalc (1 mentions)
5 protocols using mrq 26
1
Immunohistochemical Analysis of GBS, Histone H3, and CD163
2
Quantifying macrophage subsets in FFPE tissues
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For the identification of macrophages, CD68 was utilized as a pan-macrophage marker, while CD163 served as a specific marker for M2 macrophages. IHC staining for CD68 (1:100, PG-M1, Dako Cytomation, Carpinteria, CA, USA) and CD163 (ready-to-use, MRQ-26, Cell Marque, Rocklin, CA, USA) was performed on FFPE tissue sections using the BenchMark ULTRA Slide Staining System (Ventana Medical Systems, Tucson, AZ, USA) following the manufacturer’s instructions. After IHC staining, the proportion of CD68-positive or CD163-positive areas within each sample was evaluated using previously described methods [21 (link)]. Specifically, the core area of each tissue was divided into quarters, and five fields were randomly selected from the central area and each quarter at ×200 magnification (Supplemental Fig. S2 ). Areas with tumor necrosis and fibrosis were excluded from the analysis. The Color Deconvolution2 plug-in within ImageJ version 1.53t software (National Institutes of Health, Bethesda, MD, USA) was used to separate the diaminobenzidine-positive areas. Density of macrophages was determined by calculating the average positive area (%) of five selected areas in each tissue sample.
3
Multiplex Immunohistochemistry of FFPE Samples
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Opal 7-colour kit (PerkinElmer, NEL811001KT) was used for multiplex IHC. Four micrometers of FFPE sections were dewaxed and rehydrated. In the first round antigen was retrieved with a pressure cooker (EDTA pH 8.0) at 125 °C for 3 min. Slides were cooled to room temperature (RT), washed with TBST/0.5% Tween (3 times, 5 min) and incubated with H2O2 (3%) for 10 min. Slides were washed and blocked with blocking buffer (Dako, Glostrup, Denmark) for 10 min. Primary antibody, CD163 (Cell Marque, MRQ-26, 1:500, dye540), was incubated at RT for 30 min. Slides were washed and an HRP-conjugated secondary antibody was incubated at RT for 10 min. TSA dye (1:50) was applied for 10 min after washes. This was repeated five more times using the following antibodies, CD68 (Leica Biosystems, 514H12, 1:100, dye570), CD206 (Abcam, Ab64693, 1:6000, dye620), IRF8 (Santa Cruz, E-9, 1:3000, dye650), PDL1 (Spring Bioscience, SP142, 1:2000, dye520), and multi-cytokeratin (Leica Biosystems, NCL-L-AE1/AE3, 1:200, dye690). For second and subsequent rounds antigen retrieval was performed in EDTA (pH 8.0) buffer using a microwave (100–150 mW, 15 min). Nuclei were stained with DAPI (PerkinElmer) and mounted with medium (HardSet, Vectashield). Secondary antibodies anti-rabbit (PerkinElmer, NEF812001EA) or anti-mouse (PerkinElmer, NEF822001EA) were used at a 1:1000 dilution.
4
Comprehensive Immunohistochemical Analysis of FFPE Samples
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A TMA of 168 samples was immunohistochemically analyzed for HLA class I expression with the following antibodies: HCA2 (HLA-A), HC10 (HLA-B/C; both provided by Prof. Neefjes of our institute), anti-beta-2-microglobulin (β2m; DAKO, Denmark), MEM-E/02 (HLA-E; Bio-Rad, USA), and 4H84 (HLA-G; from BD Pharmingen, USA) (9 (link)). PD-L1 was determined on 213 whole-mount sections using the E1L3N clone (Cell Signaling, USA) (11 (link)).
Whole-mount sections from 213 FFPE tissue blocks were immunohistochemically stained for CD8 (C8/144B, DAKO, Denmark), FoxP3 (236A/E7, AbCam, England), and CD163 (MRQ-26, Cell Marque, Rocklin, USA) using the Ventana protocol and autostainer with heat induced antigen retrieval. Details of different IHC stainings are summarized in Table S1 in Supplementary Material.
Whole-mount sections from 213 FFPE tissue blocks were immunohistochemically stained for CD8 (C8/144B, DAKO, Denmark), FoxP3 (236A/E7, AbCam, England), and CD163 (MRQ-26, Cell Marque, Rocklin, USA) using the Ventana protocol and autostainer with heat induced antigen retrieval. Details of different IHC stainings are summarized in Table S1 in Supplementary Material.
5
CD163 Immunohistochemistry in Liver Biopsies
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Immunohistochemical analyses were performed in all biopsies using a mouse monoclonal antihuman CD163 antibody (Cell Marque, Rocklin, CA,USA; MRQ-26). Cell clustering in liver tissues was quantified as previously reported. 13 Statistical analysis. Data are reported as mean ± standard deviation (SD) for continuous normally distributed variables, as median and interquartile range for continuous non-normally distributed variables and number and frequency (%) for categorical variables. Comparison between more than two groups was performed with Kruskal-Wallis test for continuous not normally distributed variable and by ANOVA for continuous normally distributed variables. Similarly, comparison between two groups was performed by Mann-Whitney test for not normally distributed variables and by t-test for normally distributed variables. For categorical data, the Fisher exact test or χ 2 test was used as appropriate. Spearman or Pearson correlation was used as appropriate to evaluate the correlation between sCD163 levels with anthropometric, metabolic and histological features. Multivariable regression analysis was performed to assess the association between sCD163 levels and glucose and lipid fluxes from tracer studies (rp). Values of P < 0.05 were considered statistically significant. All calculations were performed with MedCalc Software bvba version 12 (Mariakerke, Belgium).
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