Tumoral and peritumoral specimens from ten patients paired with CTC scRNA-seq data were formalin-fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin for histopathological evaluation at the pathology department of Zhongshan Hospital. IHC studies employed 5-mm sections of formalin-fixed, paraffin-embedded tissue. All were stained on the Leica Bond III automated platform using the Leica Refine detection kit (Leica Microsystems, Wetzlar, Germany). Sections were deparaffinized, and heat-induced epitope retrieval was performed on the unit using EDTA for 20 min at 90 °C. Sections were incubated for 30 min with primary antibody FoxP3 (Cat# ab20034, diluted 1:250, Abcam) and CCL5 (Cat# AF-278-NA, diluted 1:250, R&D Systems). The EnVision G/2 Double stain system (DAKO) was used for dual-color antigen staining. All tissues were counterstained with hematoxylin. Isotype controls used were rabbit immunoglobulin fraction and mouse IgG1 from DAKO. Whole tissue sections were imaged with a LeicaSCN400 histology scanner. All sections were evaluated by two pathologists without knowing patient clinical characteristics and outcomes.
Leica bond 3
Manufactured by Leica Microsystems
Sourced in Germany, United States, Australia, Japan, United Kingdom
The Leica Bond III is an automated slide staining system designed for immunohistochemistry and in situ hybridization applications in pathology laboratories. It provides consistent and reliable staining results through precise control of reagent application, incubation times, and reaction temperatures.
Automatically generated - may contain errors
Lab products found in correlation
Bond polymer refine detection kit, by Leica Microsystems (3 mentions)
Cts b clone d1c7y, by Cell Signaling Technology (2 mentions)
Cts k clone 3d9, by Abcam (2 mentions)
Cts l 5 k h clone epr8011, by Abcam (2 mentions)
Cts s clone epr5128, by Abcam (2 mentions)
Mouse igg1, by Agilent Technologies (1 mentions)
Envision g 2 doublestain system, by Agilent Technologies (1 mentions)
Foxp3, by Abcam (1 mentions)
Bap1 antibody, by Santa Cruz Biotechnology (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Active caspase 3, by Cell Signaling Technology (1 mentions)
Is600, by Agilent Technologies (1 mentions)
Ki 67 mib 1, by Agilent Technologies (1 mentions)
Hpa002643, by Atlas Antibodies (1 mentions)
Canada balsam, by Merck Group (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Vectastain abc, by Vector Laboratories (1 mentions)
Cd11c, by GeneTex (1 mentions)
Cd209, by BD (1 mentions)
Monoclonal mouse anti human cd8 antibody, by Agilent Technologies (1 mentions)
29 protocols using leica bond 3
1
Immunohistochemical Analysis of Tumor Microenvironment
2
Immunohistochemical Evaluation of BAP1 Expression
3
Immunohistochemical Evaluation of HER2, ER, PgR, and Ki67 in Solid Cancers
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IHC for HER2 was performed on 4-µm thick FFPE whole-tissue sections using the PATHWAY anti-HER-2/neu rabbit monoclonal antibody (clone 4B5, Ventana Medical Systems, Tokyo, Japan) on the Leica BOND-III (Leica Microsystems, Wetzlar, Germany). External positive controls were tested with each run. To determine the level of HER2 expression, the membrane staining pattern was estimated and scored on a scale of 0 to 3 + for breast15 and gastric16 cancers. An established breast cancer HER2 IHC scoring method [15 (link)] was applied to other solid cancers including lung, bladder, renal pelvic, cervical, endometrial, ovary, and prostate cancers, uterine sarcoma, and extra-mammary Paget’s disease; for gastric cancers and cancers with clear luminal structure (colorectal, gallbladder, and fistula cancers), the gastric cancer scoring system [16 (link)] was used. Scoring was performed independently by three pathologists. The IHC for the ER and PgR was performed using the CONFIRM rabbit monoclonal antibodies (clone SP1 and IE2, respectively). Tumors with ≥ 1% of cells showing positive nuclear staining for the expression of ER and PgR were evaluated as ER/PgR-positive. IHC for Ki67 was performed using a mouse monoclonal anti-human Ki67 antibody (MIB-1, Dako). The labeling index (LI) was assessed as the percentage of tumor cells showing definite nuclear staining among > 1000 invasive tumor cells.
4
Immunohistochemical Staining of FFPE Tissues
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Antibodies were chosen based on their suitability for use on FFPE tissue and optimised. All staining was performed on a Leica Bond III automated immunostainer (Leica Microsystems, Wetzlar, Germany). The following primary antibodies were used: ProCaspase-3 (1 : 200; Rabbit polyclonal, HPA002643, Atlas Antibodies, Stockholm, Sweden), Active Caspase-3 (1:750; Rabbit polyclonal, 9661, CST, Cell Signaling Technology, Danvers, MA, USA), COX-2 (1 : 400; Mouse monoclonal; CAY160112-1, Cayman (@Cambridge Bioscience, Cambridge, UK)), and Ki-67 (MIB1) (1 : 100; Dako, Glostrup, Denmark, M7240). Negative controls for both rabbit and mouse antibodies (Negative Control Mouse Cocktail from mouse IgG1, IgG2a, IgG2b, IgG3, and IgM, IS750, Dako; Negative Control Rabbit Immunoglobulin fraction of serum from non-immunized rabbits, IS600, Dako) were included; and no staining was observed in these controls. Visualisation was performed using the enhanced diaminobenzidine tetrachloride with Harris′ haematoxylin as counterstain.
5
Immunohistochemistry on FFPE Tissues
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Immunohistochemistry was performed on whole formalin-fixed, paraffin-embedded (FFPE) sections on both lymph node and bone marrow tissues. Reactions were performed on Leica Bond-III (Leica Microsystems, IL, USA). CD20 (monoclonal mouse anti-human CD20, clone L26, Dako) antibody was diluted 1:100 with 20 min heat retrieval in 9.0 pH buffer, while the β-catenin (ready to use primary β-catenin antibody, clone 17C2, Leica) antibody was pre-diluted with 20 min heat retrieval in 6.9 pH buffer.
6
Immunohistochemical Analysis of MUC1 and MUC2 in Tumor Samples
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All samples were stained with both anti-MUC1 (Ma695, 1:200 dilution) and anti-MUC2 (Ccp58, 1:100 dilution). Both antibodies were obtained from Leica Microsystems (Wetzlar, Germany). IHC staining was performed using a Leica BOND III automatic immunostainer (Leica Microsystems) after incubation of the sample in a decloaking chamber for antigen activation. All the stained samples were analyzed by the same pathologist (HY). The samples were scored according to the percentage of tumor cells that were stained and were arbitrarily defined as being IHC-positive if more than 30% of tumor cells were positively stained29 (link) (Figs. 3 c, d, 4 c, d, 5 c, d).
7
MPM Tissue Sectioning and Immunohistochemistry
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MPM cell lines were cultured into a 3D spheroid model. Cells were spun down and embedded into cell blocks that were further processed into paraffin blocks. MPM tissue blocks and cell blocks were sectioned at 0.4 μm thickness, deparaffinised, and rehydrated in graded concentrations of xylene and ethanol. Antigen retrieval and immunohistochemical staining were performed on an automated Leica Bond III (Leica Microsystems, Melbourne, Australia) as previously described (21 (link)). IHC stained sections were imaged with an Axio Imager.M2 (ZEISS).
8
Immunohistochemical Analysis of CK5 in Tumors
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The biopsy fragments were placed into buffered formalin (10%) for 48 h and subsequently paraffin was added. All stages of the immunohistochemical technique were facilitated by the use of an automated immunohistochemistry instrument (Leica Bond-III; Leica Microsystems, GmbH, Wetzlar, Germany), according to the manufacturer’s instructions and using a primary monoclonal mouse antibody specific for CK5 (clone XM26, ready to use; Novocastra Laboratories Ltd., Newcastle upon Tyne, UK). Following dehydration in pure alcohol, the sections were placed in benzene to replace ethanol prior to embedding in paraffin and subsequently fitted using Canada balsam (Sigma-Aldrich, St. Louis, MO, USA). The microscopic evaluation was performed with a Nikon Eclipse E600 microscope (Nikon Corporation, Tokyo, Japan) and the images were obtained using the Laboratory Universal Computer Image Analysis G system (Laboratory Imaging Co., Prague, Czech Republic). The immunohistochemistry for K5 in the tumor cells was evaluated according to the following scores: 0 (0% CK5-positive cells), 1 (<10% CK5-positive cells), 2 (10–30% CK5-positive cells) and 3 (>30% CK5-positive cells).
9
Histological and Immunohistochemical Analysis of Cardiac Tissue
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The ventricular tissue was fixed in formalin and embedded in paraffin using standard histological procedures. The tissue was cut to yield 5‐μm‐thick cross sections. The sections were subsequently stained with hematoxylin and eosin (HE) and Masson's trichrome staining to determine the extent of fibrosis.
Immunohistochemical examinations were performed on 5‐μm‐thick formalin‐fixed and paraffin‐embedded tissue sections. All steps were performed on a Leica Bond III automated system (Leica Microsystems) according to the manufacturer's instructions. In brief, specimens were deparaffinized and antigen was retrieved on the instrument. All slides were incubated with primary antibodies against CD68 (diluted 1:1000; Dako), CD209 (1:1000; BD Pharmingen), or CD11c (1:100; GeneTex) for 16 min, followed by incubation with a mouse‐rabbit‐horseradish peroxidase polymer and 3,3′‐diaminobenzidine substrate. The sections were then incubated in primer (anti‐rabbit and anti‐mouse) for 8 minutes. Antibody binding was visualized using the avidin‐biotin complex method according to the manufacturer's instructions (Vectastain ABC; Vector). The primary antibody was omitted from these protocols as a negative control. The sections were subsequently counterstained with HE.
Immunohistochemical examinations were performed on 5‐μm‐thick formalin‐fixed and paraffin‐embedded tissue sections. All steps were performed on a Leica Bond III automated system (Leica Microsystems) according to the manufacturer's instructions. In brief, specimens were deparaffinized and antigen was retrieved on the instrument. All slides were incubated with primary antibodies against CD68 (diluted 1:1000; Dako), CD209 (1:1000; BD Pharmingen), or CD11c (1:100; GeneTex) for 16 min, followed by incubation with a mouse‐rabbit‐horseradish peroxidase polymer and 3,3′‐diaminobenzidine substrate. The sections were then incubated in primer (anti‐rabbit and anti‐mouse) for 8 minutes. Antibody binding was visualized using the avidin‐biotin complex method according to the manufacturer's instructions (Vectastain ABC; Vector). The primary antibody was omitted from these protocols as a negative control. The sections were subsequently counterstained with HE.
10
FISH Analysis of Cancer Biomarkers
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After deparaffinization and hydration of 3–4 µm-thick slides, sections were further processed for FISH according to our in-house protocol. In brief, the slides were pretreated automatically with the Leica Bond-III (Leica Microsystems, Wetzlar, Germany), then manually washed with water and dehydrated by 70%, 80%, and 100% ethanol. Subsequently, slides were incubated overnight with commercially available SPEC CDKN2A/CEN 9 and SPEC RB1/13q12 Dual Color Probe kits (ZytoVision, Bremerhaven, Germany) as well as LSI EGFR SpectrumOrange/CEP7 SpectrumGreen (Abbott, Chicago, IL, USA) probes.
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