Formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks were cut with a regular microtome for further immunohistochemical and methylation analysis. Immunohistochemistry for Ki67 was performed by an automated slide processing system (Autostainer Plus Link, Dako, Glostrup, Denmark; MIB-1/clone M7240, Dako) as previously described [8 (link)]. The Ki67 proliferation index was obtained by counting 500 cells in the most densely stained area and is given as a percentage (0–100%). Methylation analysis was performed of samples with resection at recurrence and differing histological features as compared to the first resection. DNA extracted from FFPE tissue was analyzed as described previously using Illumina Infinium HumanMethylation850 BeadChip (850 k) arrays (Illumina, San Diego, CA, USA) [9 (link)]. Samples were classified using the previously published MolecularNeuropathology classifier [10 (link)].
Autostainer plus link
Manufactured by Agilent Technologies
Sourced in Denmark
The Autostainer Plus Link is a fully automated slide staining system designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. It provides consistent and reproducible staining results by automating the staining process, including reagent handling, incubation, and washing steps.
Automatically generated - may contain errors
Lab products found in correlation
Mib 1 clone m7240, by Agilent Technologies (2 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions)
Ndp view2, by Hamamatsu Photonics (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Clone d5 16 b4, by Agilent Technologies (1 mentions)
Clone d38b1, by Cell Signaling Technology (1 mentions)
Clone vim3b4, by Agilent Technologies (1 mentions)
Anti egfr monoclonal antibody, by Cell Signaling Technology (1 mentions)
Envision, by Agilent Technologies (1 mentions)
5 protocols using autostainer plus link
1
Comprehensive Characterization of Recurrent Glioma
2
Characterization of Liver Malignant Lesions
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Five-µm liver sections were colored with the Masson’s Trichrome (MT) stain. Tissue sections were also processed for immunohistological analysis with antibodies against Melan-A (clone A103, Agilent Dako, Mississauga, ON, Canada) and αSMA (clone 1A4, Agilent Dako) in a Dako Autostainer Plus Link, according to the manufacturer’s protocol using the EnVision peroxidase procedure with the DAB or Magenta chromogen (Agilent Dako) [45 (link)]. The demasking was done at high pH. Secondary antibody without primary antibody was used as negative control, and positive controls were performed with skin and muscle tissues. Sections were digitized with a slide scanner (NanoZoomer 2.0HT; Hamamatsu Photonics, Bridgewater, NJ, USA), and images were analyzed with the NDP.view 2 software (Hamamatsu Photonics). Liver malignant lesions were counted on MT-stained slides using the measure feature of NDP.view 2. They were then categorized according to their size (<50 µm, 50–500 µm, >500 µm in diameter) [23 (link)].
3
Immunohistochemical Analysis of Protein Markers
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Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue TMA sections. Briefly, 3 μm-thick TMA tissue sections were placed onto adhesive slides and treated with the PT link (DAKO) solution. Immunohistochemical staining was performed using the following primary antibodies: anti-Fatty Acid Synthase polyclonal antibody (1:100, Enzo Life Sciences), anti-EGFR monoclonal antibody (1:100, clone D38B1, CellSignaling), anti-Cytokeratin 5/6 monoclonal antibody (1:50/100, Clone D5/16 B4, DAKO) anti Monoclonal Mouse Anti-vimentin (1:100/200, Clone Vim 3B4, DAKO). Sections were washed with PBS and sequentially incubated at room temperature for 45 minutes with antirabbit or antimouse IgG. Immunodetection was performed with the kit EnVision™ (DAKO, Glostrup, Denmark) using the AutostainerPlus Link (DAKO).
4
Retrospective Study of Glioma Patients
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This retrospective study was approved by the local Ethics Committee. All investigated tumor specimens were acquired from clinical samples obtained by resection or biopsy procedures during the diagnostic workup of 122 glioma patients (67 men, 55 women, mean age: 45, range 18–84 years) diagnosed between 2000 and 2014 without any prior glioma specific therapy. Primary evaluation of all tumor samples was performed prior to the implementation of the current WHO 2016 classification and therefore samples were classified based on the WHO classification 2007 and complimented with additional immunostaining of the isocitrate dehydrogenase 1 R132H (i.e., IDH1-R132H) mutational status as described recently [22 (link)]. Immunohistochemistry for Ki67 was performed by an automated slide processing system (Autostainer Plus Link, Dako, Glostrup, Denmark; MIB-1/clone M7240, Dako). The Ki67 proliferation index was determined by counting 500 cells in the most densely stained area and was expressed as percentage (0–100%). A subpopulation was also re-evaluated according to the current WHO 2016 classification. Patients’ characteristics are given in Table 1 . Moreover, from each patient, preoperative and pretreatment MET PET data was available. The study population analyzed has already been described in detail in a previous investigation with different purpose, analysis and results [22 (link)].
5
Immunohistochemical Analysis of Tissue Samples
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Formalin-fixed paraffin-embedded tissue samples were cut into 3-μm slices and mounted on positively charged glass slides. Immunohistochemistry was performed using a Dako Autostainer Plus-Link (Dako, Glostrup, Denmark). Details of the antibodies and staining protocols used are listed in Supplementary Table 2 .
For all antibodies, specific staining patterns [i.e., nuclear for KI67, TTF-1, membranous for CD56, GLUT1, PD-L1 (tumor cells, macrophages, stroma) and cytoplasmic for CD 44, Chromogranin, PD-1] were evaluated. Any intensity was recorded. TTF-1, CD56, Chromogranin A, and Synaptophysin were classified as positive if the majority of tumor cells showed specific staining reactivity.
For CD44, GLUT-1, and HIF-1a, H-scores were calculated according to the following formula (26 (link), 27 (link)):
[1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)].
All viable tumor cells within the biopsy were taken into account when applying the semi-quantitative scoring system.
For all antibodies, specific staining patterns [i.e., nuclear for KI67, TTF-1, membranous for CD56, GLUT1, PD-L1 (tumor cells, macrophages, stroma) and cytoplasmic for CD 44, Chromogranin, PD-1] were evaluated. Any intensity was recorded. TTF-1, CD56, Chromogranin A, and Synaptophysin were classified as positive if the majority of tumor cells showed specific staining reactivity.
For CD44, GLUT-1, and HIF-1a, H-scores were calculated according to the following formula (26 (link), 27 (link)):
[1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)].
All viable tumor cells within the biopsy were taken into account when applying the semi-quantitative scoring system.
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