After biopsy, tissues were routinely prepared, fixed in 4% neutral buffered formalin, embedded in paraffin and cut to 3-µm thickness. Standard haematoxylin-eosin and Von Kossa staining were performed. Immunoperoxidase staining for antibodies against various bone and calcification markers was performed on an automated DAKO autostainer platform (Agilent Technologies, Santa Clara, CA, USA) with standard staining protocols for all markers. A list of all markers, company information and catalogue numbers are given in Appendix S1 .
Dako autostainer platform
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The DAKO autostainer platform is a fully automated system designed for immunohistochemistry (IHC) and in situ hybridization (ISH) sample preparation. The platform automates the staining process, including reagent dispensing, incubation, and washing steps, to provide consistent and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Clone d5 16b4, by Roche (1 mentions)
Csa 2, by Agilent Technologies (1 mentions)
Rabbit anti tnfr2, by Merck Group (1 mentions)
Mouse anti neurofilament nf, by Merck Group (1 mentions)
Envision system horse radish peroxidase labelled polymer, by Agilent Technologies (1 mentions)
Mouse anti cd68 clone pg m1, by Agilent Technologies (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
4 protocols using dako autostainer platform
1
Histological Assessment of Bone Calcification
2
Immunohistochemical Analysis of Gynecomastia
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Immunohistochemical (IHC) staining was performed on 22 cases with gynaecomastoid change. Commercially available antibodies used were: anti-oestrogen receptor (ER) (clone SP1, 1:50 dilution; Cell Marque, Rocklin, CA, USA), anti-progesterone receptor (PR) (clone Y85, 1:40 dilution; Cell Marque), and anti-androgen receptor (AR) (clone AR441, 1:100 dilution; Thermo-Scientific, Fremont, CA, USA). IHC staining for cytokeratin (CK) 5/6 was performed on selected cases (clone D5/16B4; Ventana Medical Systems, Tucson, AZ, USA). Staining was performed on the Dako Autostainer platform (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer's instructions, with appropriate controls.
Oestrogen receptor, PR and AR staining in gynaecomastoid change was scored by a breast pathologist (J.M.J.), and reported via the Allred scoring system, with intensity scores (negative, 0; weak, 1; intermediate, 2; strong, 3) and proportion scores (none positive, 0; ≤1%, 1; 1-10%, 2; 11-33%, 3; 34-66%, 4; 67-100%, 5) being added to generate a sum score (with scores of 0-2 being interpreted as negative and 3-8 as positive. Background uninvolved (non-gynaecomastoid) glands served as internal controls.
Oestrogen receptor, PR and AR staining in gynaecomastoid change was scored by a breast pathologist (J.M.J.), and reported via the Allred scoring system, with intensity scores (negative, 0; weak, 1; intermediate, 2; strong, 3) and proportion scores (none positive, 0; ≤1%, 1; 1-10%, 2; 11-33%, 3; 34-66%, 4; 67-100%, 5) being added to generate a sum score (with scores of 0-2 being interpreted as negative and 3-8 as positive. Background uninvolved (non-gynaecomastoid) glands served as internal controls.
3
Gastric Histology and H. pylori Analysis
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Standard H&E staining was performed on 3 μm sections of all FFPE blocks of the cases and controls on the Dako autostainer platform (Dako, Denmark). Joint histologic review of all the cases and controls using a consensus approach by two registered pathologists and one resident was performed. Gastric cancer was classified using the Lauren classification, while gastritis using the Sydney system. The presence of H. pylori was assessed on newly stained H&E and original Giemsa sections, and was reported as positive if identified on either stain.
4
Immunohistochemical Staining of Inflammatory Markers
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Immunohistochemical staining was performed using the Dako autostainer platform (Dako, Denmark) as previously described [24 (link)]. Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems). The antigen-antibody complex was visualized using EnVision+System horse-radish peroxidase-labelled Polymer (Dako), PowerVision+Poly-HRP IHC (AH Diagnostics), or CSAII (Dako) detection systems.
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