Bond rx automated stainer

Manufactured by Leica

Sourced in Germany, United Kingdom

The Leica Bond Rx Automated Stainer is a laboratory equipment designed for automated staining of tissue samples. It automates the process of applying various stains to specimen slides, ensuring consistent and reproducible results.

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Lab products found in correlation

Bond dewax solution, by Leica (4 mentions) Bond epitope retrieval solution 1, by Leica (3 mentions) Bond epitope retrieval 1, by Leica (3 mentions) Bond refine detection kit, by Leica (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Opal 6 plex detection kit, by Akoya Biosciences (2 mentions) Vectra3 microscope, by Akoya Biosciences (2 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions) Phenochart whole slide viewer, by Akoya Biosciences (2 mentions) Inform automated image analysis software, by Akoya Biosciences (2 mentions) Epitope retrieval buffer 2, by Leica (1 mentions) Imagescope software, by Leica (1 mentions) Aperio scanscope slide scanner, by Leica (1 mentions) Vectra3 slide scanner, by PerkinElmer (1 mentions) Bond plus slides, by Leica (1 mentions) Anti pd 1, by Abcam (1 mentions) Anti cd8, by Abcam (1 mentions) Anti pd l1, by Abcam (1 mentions) Bond antibody diluent buffer, by Leica (1 mentions) Anti cd3, by Abcam (1 mentions)

19 protocols using bond rx automated stainer

Immunohistochemical Analysis of Mammary Tumors

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Treated and control mammary tumors were fixed in 10% formalin for 48 h and then transferred to 70% ethanol until histological processing. Tumor sections were processed for Hematoxyilin–Eosin staining or immunohistochemistry using antibodies listed in Supplementary Table 1. All the antibodies used in this study were validated by the supplier as well as independent publications from academic laboratories. However, before we use any of the antibodies, we have validated their suitability by titrating the amount of antibody and running positive and negative controls. All antibodies were stained on the Leica Bond RX Automated Stainer with either Epitope Retrieval Buffer 1 (ER1), Epitope Retrieval Buffer 2 (ER2), or with Enzyme 1 (Leica). All antibodies were diluted in Power Vision Super blocker (Leica). The IHC slides were scanned using Aperio ScanScope slide scanner (Aperio Technologies, CA), and the images were analyzed using Positivity Pixel Count 9.0 algorithm within ImageScope software (Aperio Technologies, CA) as described previously^{75 (link)}.

Workenhe S.T., Nguyen A., Bakhshinyan D., Wei J., Hare D.N., MacNeill K.L., Wan Y., Oberst A., Bramson J.L., Nasir J.A., Vito A., El-Sayes N., Singh S.K., McArthur A.G, & Mossman K.L. (2020). De novo necroptosis creates an inflammatory environment mediating tumor susceptibility to immune checkpoint inhibitors. Communications Biology, 3, 645.

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Automated HER2 Immunohistochemistry Assay

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After topical tissue staining and DDSI was completed, each specimen was placed in 10% formalin (Biochemical Science Inc, Swedesboro, NJ) for at least 24 hrs and then prepared for IHC staining of HER2. Four micron slices were cut and mounted on Leica Bond Plus Slides (Cat # 00270) and air-dried at room temperature. Using the automated protocol of the Leica Bond Rx Automated Stainer (Leica Products/Equipment, Leica Microsystems, Inc., Buffalo Groove, IL), the slides were baked for 30 minutes and dewaxed with Leica Bond Dewax solution (Cat #AR9222). The antigen retrieval was Bond Epitope Retrieval 2 (Cat #ar9640), carried out in a pH 9.0 solution for 20 minutes. The HER2 primary antibody dilution was 1:300 for 15 minutes (Abcam Cat # ab16901; Abcam Inc., Cambridge, MA). Primary antibody binding was visualized using Leica Bond Refine Detection kit (Cat # DS9800) with a diaminobenzidine (DAB) chromogen and a hematoxylin counterstain. Bright field, whole fit images of stained tissue (H&E and HER2 IHC) were scanned using the PerkinElmer Vectra3 slide scanner.

Meng B., Folaron M.R., Byrd B.K., Samkoe K.S., Strawbridge R.S., Barth C., Gibbs S.L, & Davis S.C. (2020). Topical dual-probe staining using quantum dot-labeled antibodies for identifying tumor biomarkers in fresh specimens. PLoS ONE, 15(3), e0230267.

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Immunohistochemical Analysis of Tumor-Infiltrating Lymphocytes and Immune Checkpoint Proteins

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IHC was conducted on 1 µm thick full tissue sections and stained with markers for tumor-infiltrating lymphocytes (TILs; CD3, CD8) and immune checkpoint proteins (PD-1, PD-L1). Staining was performed using a Leica Bond RX Automated Stainer (Leica Products/Equipment, Leica Microsystems, Inc., Buffalo Groove, IL). Slides with incubated for 30 minutes at 95°C, followed by dewaxing with Leica Bond Dewax solution (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. AR9222), antigen retrieval with Bond Epitope Retrieval 1 solution (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. AR9961) and blocking of unspecific binding sites with 2% goat serum. Primary antibody binding was visualized with diaminobenzidine chromogen and a hematoxylin counterstain, using the Leica Bond Refine Detection kit (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. DS9800). Primary antibodies were diluted in Leica Bond Antibody Diluent buffer (Leica Biosystems, Inc., Buffalo Groove, IL; Cat no. AR9352) as follows: anti-CD3 (Abcam, Cambridge, MA, USA; Cat no. ab5690; rabbit polyclonal) 1:300, anti-CD8 (Abcam, Cambridge, MA, USA; Cat no. ab4055; rabbit polyclonal) 1:300), anti-PD-1 (Abcam, Cambridge, MA, USA; Cat no. ab137132; rabbit polyclonal) 1:75, anti-PD-L1 (Abcam, Cambridge, MA, USA; Cat no. ab205921; rabbit polyclonal) 1:100).

Gruber E.S., Oberhuber G., Pils D., Stork T., Sinn K., Gruber S., Nica R., Kolmer D., Turner S.D., Schlederer M., Widder J., Doerr W., Teleky B, & Kenner L. (2020). The Determination of Immunomodulation and Its Impact on Survival of Rectal Cancer Patients Depends on the Area Comprising a Tissue Microarray. Cancers, 12(3), 563.

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Immunohistochemical Analysis of γ-H2AX in Tumor Tissue

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Tumor tissue was fixed in 10% neutral buffered formalin and embedded in paraffin, before being sectioned (4 μM thick) and stained using the Bond RX Automated Stainer (Leica Biosystems). Heat induced antigen retrieval was performed at pH 9 (Bond Epitope Retrieval solution 2, Leica Biosystems), 100 °C for 30 min, before Υ-H2AX antibody incubation (1:500 dilution, Cell Signaling, Clone 20E3) for 60 min. Detection was performed with diaminobenzidine (Bond Polymer Refine Detection, Leica Biosystems) and slides were counterstained with haematoxylin. Slides were imaged using a slide scanner (AperioCS2, Leica Biosystems), and data were analyzed using QuPath^{52 (link)} as described^{53 (link)}.

Aziz D., Portman N., Fernandez K.J., Lee C., Alexandrou S., Llop-Guevara A., Phan Z., Yong A., Wilkinson A., Sergio C.M., Ferraro D., Etemadmoghadam D., Bowtell D.D., Serra V., Waring P., Lim E, & Caldon C.E. (2021). Synergistic targeting of BRCA1 mutated breast cancers with PARP and CDK2 inhibition. NPJ Breast Cancer, 7, 111.

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Ki-67 Analysis of NCI-237-R PDX Tumors

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NCI-237-R^PDX tumor fragments from vehicle- and NCGC00420737-treated animals were fixed in 10% neutral-buffered formalin and incubated at 4°C for 24 hours with gentle shaking. Fixed tumors were submitted to the UT Southwestern Histo Pathology Core for paraffin embedding and sectioning. Hematoxylin- and eosin-staining was performed by the UT Southwestern Histo Pathology Core. Ki-67 immunohistochemical staining was performed by the UT Southwestern Simmons Comprehensive Cancer Center Tissue Management Shared Resource using a BOND-RX Automated Stainer (Leica Biosystems) and human-specific anti-Ki-67 antibody (Cell Signaling Technology #9027, RRID: AB_2636984, 1:400 dilution). Whole-slide scans were captured at 40X magnification using a Vectra Polaris slide scanner (Akoya Biosciences) and images were generated using Phenochart^™ 1.1.0 software (Akoya Biosciences).

Salzmann M., Pervushin K., Wider G., Senn H, & Wüthrich K. (1998). TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13585-13590.

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Immunohistochemical Staining of p53β and CA9

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For immunohistochemical (IHC) staining, 4‐μm sections from paraffin‐embedded tissues were used. The KJC8 antibody towards p53β was generously provided by the Bourdon Laboratory (University of Dundee, Ninewells Hospital and Medical School, Dundee, UK) and was detected using EnVision Dual Link (Dako, Glostrup, Denmark) and diaminobenzidine chromogen (DAB), with DAB enhancer (Leica Biosystems, Wetzlar, Germany). The CA9 antibody (MRQ‐54; Cell Marque, Rocklin, CA, USA) was used to detect CA9‐positive cells using an automated IHC method (BOND RX automated stainer; Leica Biosystems, Wetzlar, Germany).

Kazantseva M., Eiholzer R.A., Mehta S., Taha A., Bowie S., Roth I., Zhou J., Joruiz S.M., Royds J.A., Hung N.A., Slatter T.L, & Braithwaite A.W. (2018). Elevation of the TP53 isoform Δ133p53β in glioblastomas: an alternative to mutant p53 in promoting tumor development. The Journal of Pathology, 246(1), 77-88.

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Spatial Expression of Gli1 and Atoh1 mRNA

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To assess Gli1 mRNA expression in the cerebellum, we used the RNAscope LS Multiplex Assay (Advanced Cell Diagnostics). P11 mouse brain sections were baked at 65 °C for 45 min, post-fixed in 4% paraformaldehyde for 15 min, and dehydrated prior to the assay. Multiplexed single-molecule fluorescent in situ hybridization (smFISH) against Gli1 and Atoh1 mRNA was performed on the BOND RX automated stainer (Leica) using the RNAscope reagents. The Gli1 probe consists of 20 double-Z probes targeting nucleotides 25–1025 on the mouse transcript NM_010296.2 and was developed with the fluorophore Opal 520 (Perkin Elmer). The Atoh1 probe consists of 20 double-Z probes targeting nucleotides 847–2088 on the mouse transcript NM_007500.4 and was developed in Opal 570.

Nguyen V., Sabeur K., Maltepe E., Ameri K., Bayraktar O, & Rowitch D.H. (2017). Sonic Hedgehog Agonist Protects Against Complex Neonatal Cerebellar Injury. Cerebellum (London, England), 17(2), 213-227.

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Immunohistochemical Analysis of Aortic Valve pERK

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Immunohistochemical staining of aortic valves was performed with a Leica BondRX automated stainer, according to the manufacturer instructions. In brief, tissues for immunostaining underwent deparaffinization online followed by epitope retrieval for 20 min at 100°C with Leica Biosystems ER1 solution (pH6,0 #AR9961) and endogenous peroxidase activity blocking with H₂O₂. Sections were incubated with primary antibodies against pERK (Cell Signaling Technologies # 4376S; clone 20G11) at a dilution of 1:300 for 30 min at room temperature. Primary antibodies were detected with anti-rabbit HRP-conjugated polymer and 3,3′-diaminobenzidine (DAB) substrate provided in the Leica BOND Polymer Refine Detection System (#DS9800). Following counter-staining with haematoxylin, slides were scanned at 20X on a Hamamatzu Nanozoomer 2.0HT whole slide scanner and the image files uploaded to the NYUGSoM’s OMERO Plus image data management system (Glencoe Software).

Kirby M., Denihan A., Bruce I., Radic A., Coakley D, & Lawlor B.A. (1999). Influence of symptoms of anxiety on treatment of depression in later life in primary care: questionnaire survey. BMJ : British Medical Journal, 318(7183), 579-580.

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Multiplex Immunofluorescence Imaging of Tumor Tissues

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Tissue-based cyclic immunofluorescence (CyCIF) was carried out as previously described⁵⁴. Briefly, tumors were fixed in formalin and embedded in paraffin. 5 μm sections were cut and mounted on positively charged glass slides. Each tumor section underwent dewaxing and antigen retrieval on Bond Rx automated stainer (Leica). In the first round, sections were incubated with unconjugated antibodies and then labelled with secondary fluorophores. Sections then underwent sequential incubation with fluorophore-conjugated antibodies. Antibodies and dilutions used can be found in Supplementary Information Table S15. In each round, antibodies were incubated overnight followed by imaging using InCell Analyzer 2200 (GE Technologies). For each section, a region of interest was defined for the first cycle of imaging and subsequently used for every subsequent cycle without alteration. Sections were bleached before incubating with the next round of antibodies. After imaging was completed, each tile was assembled into a multi-field composite image using ASHLAR and BaSiC as previously described⁵⁴. The same sections used for tCyCIF were stained with H&E and imaged on a light microscope.

Neuhaus K., Rapposch S., Francis K.P, & Scherer S. (2000). Restart of Exponential Growth of Cold-Shocked Yersinia enterocolitica Occurs after Down-Regulation of cspA1/A2 mRNA. Journal of Bacteriology, 182(11), 3285-3288.

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Highly Multiplexed Tissue Imaging

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Tissue-based cyclic immunofluorescence (CyCIF) was carried out as previously described 54 (link) . Briefly, tumors were fixed in formalin and embedded in paraffin. 5 μm sections were cut and mounted on positively charged glass slides. Each tumor section underwent dewaxing and antigen retrieval on Bond Rx automated stainer (Leica). In the first round, sections were incubated with unconjugated antibodies and then labelled with secondary fluorophores. Sections then underwent sequential incubation with fluorophore-conjugated antibodies. Antibodies and dilutions used can be found in Supplementary Information Table S15. In each round, antibodies were incubated overnight followed by imaging using InCell Analyzer 2200 (GE Technologies). For each section, a region of interest was defined for the first cycle of imaging and subsequently used for every subsequent cycle without alteration. Sections were bleached before incubating with the next round of antibodies. After imaging was completed, each tile was assembled into a multi-field composite image using ASHLAR and BaSiC as previously described 54 (link) . The same sections used for tCyCIF were stained with H&E and imaged on a light microscope.

Bergholz J.S., Wang Q., Wang Q., Ramseier M., Prakadan S., Wang W., Fang R., Kabraji S., Zhou Q., Gray G.K., Abell-Hart K., Xie S., Guo X., Gu H., Von T., Jiang T., Tang S., Freeman G.J., Kim H.J., Shalek A.K., Roberts T.M, & Zhao J.J. (2023). PI3Kβ controls immune evasion in PTEN-deficient breast tumours. Nature, 617(7959).

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