Hematoxylin and eosin staining results of all 21 samples were reviewed by two pathologists. The specimens were fixed in formalin and embedded in paraffin before they were archived. We used the archived specimens for immunohistochemical analysis. Immunohistochemical staining was performed with Bond-Max autostainer (Leica Microsystems, Wetzlar, Germany), using CD56 (NCL-CD56-1B6, clone 1B6; 1:15; Novocastra/Leica, Newcastle-upon-Tyne, UK), chromogranin (NCL-CHROM-430, clone 5H7; 1:50; Novocastra/Leica), synaptophysin (NCL-SYNAP-299, clone 27G12; 1:50; Novocastra/Leica), and neuron-specific enolase (BBS/NC/VI-H14; 1:200; Dako, Carpinteria, CA, USA). Briefly, specimens from the paraffin-embedded blocks were cut into 5-μm sections. The sections were dewaxed in a 60°C oven, deparaffinized in xylene, rehydrated through serial dilutions of alcohol, and washed in phosphate-buffered saline (pH 7.2). Immunohistochemical staining was performed in the fully automated Bond-Max autostainer using onboard, heat-induced antigen retrieval in citrate buffer according to the ER1 protocol for 20 minutes and a VBS Refine polymer detection system (Leica Microsystems). Diaminobenzidine was used as the chromogen (Leica Microsystems). The sections were then counterstained with hematoxylin.
Bond max autostainer
Manufactured by Leica
Sourced in Germany, United States, United Kingdom, Australia, Azerbaijan
The Bond-Max autostainer is a laboratory equipment product designed for automated immunohistochemistry (IHC) and in situ hybridization (ISH) staining. It automates the staining process, including deparaffinization, rehydration, antigen retrieval, primary antibody incubation, and visualization. The Bond-Max system is capable of processing multiple slides simultaneously, ensuring consistent and reproducible results.
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Lab products found in correlation
Bond polymer refine detection kit, by Leica (13 mentions)
Bond polymer refine detection system, by Leica (4 mentions)
Ki 67 clone mib 1, by Agilent Technologies (4 mentions)
Ventana benchmark xt, by Roche (4 mentions)
Bond polymer refine kit, by Leica (3 mentions)
Cd10 clone 56c6, by Leica (3 mentions)
Bcl6 clone ln22, by Leica (3 mentions)
Cd20 clone l26, by Agilent Technologies (3 mentions)
Inform eber probe, by Roche (3 mentions)
Bond polymer kit, by Leica (3 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ab133579, by Abcam (2 mentions)
Tissue arrayer, by Beecher Instruments (2 mentions)
Bond epitope retrieval solution 2, by Leica (2 mentions)
A0485 polyclonal antibody, by Agilent Technologies (2 mentions)
Vbs refine polymer detection system, by Leica (2 mentions)
Bcl2 clone 124, by Agilent Technologies (2 mentions)
Myc clone ep121, by Cell Marque (2 mentions)
Mum1 clone ma695, by Leica (2 mentions)
117 protocols using bond max autostainer
1
Immunohistochemical Analysis of Neuroendocrine Markers
2
EBER In Situ Hybridization and IHC for HER2 and FGFR2
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To detect Epstein–Barr virus (EBV)-encoded RNA (EBER), we performed an in situ hybridization (ISH) assay as already described (Anagnostopoulos et al. 1992 (link); Fan and Gulley 2001 (link)). A ready-to-use EBER-ISH probe (BOND #PB5089) was used together with the Leica Bond-maX autostainer (Leica Biosystems, Illinois, USA) according to the standard BOND protocol. Immunohistochemical staining (IHC) was performed using sections derived from FFPE primary tissue samples with the help of the Leica Bond-maX autostainer (Leica Biosystems, Illinois, USA) according to the manufacturer’s instructions. After heat-induced epitope retrieval, the sections were incubated with anti-HER2/neu (4B5) (VENTANA—#790-4493) or anti-FGFR2 antibody (Sigma Aldrich—#HPA035305). Horseradish peroxidase-labeled anti-rabbit using the Bond Polymer Refine Detection Kit (Leica Biosystems, Illinois, United States) was employed to convert the chromogen substrate. Staining was performed with appropriate positive and negative controls. Evaluation of staining intensity was performed by a trained pathologist according to four level scoring (0—no staining, 1—light staining, 2—moderate staining, 3—strong staining) as previously described (Jia et al. 2016 (link)).
3
Immunohistochemical Staining of Tumor Samples
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As described previously [53 (link)-55 (link)], consecutive sections of 4μm thickness were cut from pre- and post-treatment tumor samples. Slides were stained with a BondMax Autostainer (Leica, Wetzlar, Germany) according to the instructions of the manufacturer. Antibodies used for IHC staining were listed in Supplementary Table 1 .
4
Renal Cancer Tissue Microarray H3K36me3 Immunostaining
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Use of human tissues in research was approved by National Research Ethics Service Committee Oxfordshire REC C (study 07/H0606/120). Nephrectomies were collected between 1995 and 2004, with informed consent from all subjects to use of tissue in ethically approved research. Formalin-fixed paraffin-embedded tumor cores were used to construct renal cancer tissue microarrays, and 4 μm sections were stained on Bondmax Autostainer (Leica). Briefly, following heat-induced epitope retrieval (20 min, pH 9) sections were stained with an antibody against H3K36me3 (Mab-183-050, Diagenode) at 1:1,000 dilution for 1 hr. Antibody binding was identified using a Polymer Detection System (Leica), followed by 3,3′-diaminobenzidine staining.
5
Immunohistochemical Evaluation of TAMs
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The cancer tissue samples were fixed with formalin and embedded with paraffin. The expression of CD68 in TAMs was examined using immunohistochemistry on 5 μm thick whole mount tissue sections, and a Leica Bond MAX auto-stainer was used. The slides were dewaxed and antigenicity was retrieved using the Leica antigen retrieval solution, then the slides were incubated with the mouse monoclonal anti-human CD68 antibody (clone KP1, ZSGB-Bio) for 30 min. The staining was visualized using a diaminobenzidine system after secondary antibody incubation. The number of membranous CD68 positive cells in the whole tumor tissue sample was calculated and at least five randomly selected high power fields (400×) were examined. Original Hematoxylin-Eosin staining slices from the pathology archive were also reanalyzed to assess the presence and degree of TAMs.
6
Immunohistochemical Analysis of Dorsal Root Ganglia
7
Prostate Tissue Sampling and Ex Vivo Culture
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Tissue sampling from radical prostatectomy specimens was performed using the PEOPLE methodology31 (link) and ex vivo culture of tissue was performed as previously described.32 (link) Briefly, gelatine sponges were placed in 24-well plates with 200 μl of RPMI supplemented with 10% (v/v) FBS 2-3 h prior to tissue harvest to allow the sponges to draw up the media. For treatment groups, 10 μM SSO was added to the RPMI. Fresh 6 mm cores were divided using a scalpel and placed on the sponges, then incubated for up to 72 h followed by harvesting for histology and RNA/protein extraction as described above. For histological analysis, samples were fixed in 10% (v/v) neutral buffered formalin and stored as FFPE blocks. In all, 4 μm sections from the FFPE blocks were stained with haematoxylin and eosin (H&E) to assess tumour content of samples as per the 100,000 Genomes Project standard operating procedures (https://www.genomicsengland.co.uk/about-genomics-england/the-100000-genomes-project/information-for-gmc-staff/sample-handling-guidance ). Slides were assessed by an experienced consultant uropathologist and tumour content was reported as 0–100% in 5% increments. For ERG immunohistochemistry, 4 μm sections from the FFPE blocks were stained on the BondMax autostainer (Leica) with anti-ERG antibody (Abcam, ab92513; 1:200 dilution).
8
Quantifying Prostate Cancer PSMA Expression
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A total of 42 (28 from PSMA-PET/CTpost group and 14 from PSMA-PET/CTpre group) patients underwent radical prostatectomy with resection of the seminal vesicles. PSMA was stained with an anti-PSMA rabbit monoclonal antibody (EPR6253, ab133579, Abcam, 1:500 dilution) on a Leica Bond-Max auto-stainer. The intensity of staining (weak, moderate or intense) and the percentage of positively stained cells (focal, regional, or diffuse) were graded as reported in a previous study [21 (link)]. Cases categorized as intense diffuse, intense regional, or moderate diffuse were considered as overexpressing PSMA protein.
9
Quantifying Ki67 Immunohistochemistry in Tissue
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Ki67 immunohistochemical labelling was performed on fresh-cut tissue sections using a 1:150 concentration of monoclonal anti-Ki67 antibody (MIB-1; Dako, Ely, UK) on a BondMax Autostainer Leica, Milton Keynes). Antigen retrieval was at Ph 9 (Bond ER2, Leica, Milton Keynes, UK) and 90 °C for 10 min. The Bond Polymer Redefine Detection kit (Leica) was used for antibody visualisation. A positive reaction was indicated by the presence of specific brown intranuclear immunoreactivity. The positive control was a canine cutaneous mast cell tumour, and this tissue without the Ki67 antibody was used as a negative control. At ×400 magnification, over an area of 2.37 mm2, the number of positively immunolabelled Ki67 cells were counted from at least 1000 cells for each sample. Ki67 expression in each sample was defined as the percentage of positive cells relative to the total number of cells counted.
10
Histological Assessment of NET Formation in ALF
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To study whether NET formation occurs in the liver parenchyma of patients with ALF, we performed histological analyses on liver tissue from 20 patients in our cohort. We specifically studied 10 patients with acetaminophen (APAP) and 10 patients with non‐APAP etiology. Liver tissue was obtained at transplantation, and after fixation in formalin, embedded in paraffin for histological assessment. Liver tissue was stained for citrullinated histones (H3citB) to assess NET formation on an automated staining machine (Bond Max autostainer; Leica, Wetzlar, Germany). We specifically used the commercially available antibody, antihistone H3 (diluted 1:100; ab5103; Abcam, Cambridge, UK). Cut tissue slides were pretreated in citrate buffer for 20 minutes. We included five liver‐tissue samples taken from otherwise discarded human donor livers obtained from another experiment (institutional ethical approval code UMCG: M14.152454) to serve as controls. Images were reviewed by an experienced liver pathologist (Y.Z.) and classified based on degree of expression of H3citB, ranging from no expression (score 0), focal expression (positive in <10% of neutrophils; score 1), moderate expression (10%‐50% of neutrophils; score 2), to extensive expression (>50% of neutrophils; score 3).
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