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Bond rxm system

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The Leica Bond RXm system is a fully automated, random-access slide staining platform designed for immunohistochemistry (IHC) and in situ hybridization (ISH) applications. The system provides consistent, reliable, and high-quality slide staining with minimal hands-on time.

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Lab products found in correlation

Asp300, by Leica (2 mentions) Osteosoft, by Merck Group (2 mentions) Polymer refine detection kit, by Leica (2 mentions) Aperio imagescope software, by Leica (1 mentions) Ab 108327, by Cell Signaling Technology (1 mentions) Imagescope software, by Leica (1 mentions) Hm355s, by Thermo Fisher Scientific (1 mentions) Ab28364, by Abcam (1 mentions) Nb100 2220, by Novus Biologicals (1 mentions) Prism 7, by GraphPad (1 mentions) Ma5 14238, by Thermo Fisher Scientific (1 mentions) Bsa pbs, by Merck Group (1 mentions) Hpa036854, by Merck Group (1 mentions) Hpa038061, by Merck Group (1 mentions) Hpa021616, by Merck Group (1 mentions) Vectra 3, by PerkinElmer (1 mentions) Opal seven color ihc kit, by PerkinElmer (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) At2 scanning system, by Leica (1 mentions) Bond polymer refine detection kit, by Leica (1 mentions)

14 protocols using bond rxm system

1

Immunohistochemical Analysis of Tumor EGFR

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Immunohistochemistry of tumor tissues derived from mice used for the imaging study was performed using a Bond RXm system (Leica, Wetzlar, Germany; all reagents from Leica) with an EGFR antibody (clone E235, 1:100, ab32077; Abcam). Briefly, slides were deparaffinized and pretreated with Epitope retrieval solution 1 (EDTA buffer [pH 6]) before the diluted primary antibody was applied for 15 min. Antibody binding was detected with a polymer refine detection kit without a post primary agent and visualized with diaminobenzidine as a dark brown precipitate. Counterstaining was done with hematoxylin. A positive control was included in each run. The stained slides were scanned with an automated slide scanner (Leica Biosystems; AT-2), and the Aperio Imagescope software (version 12.3; Leica Biosystems) was used to take representative images. The receptor expression level was evaluated by a veterinary pathologist.
Spellerberg R., Benli-Hoppe T., Kitzberger C., Berger S., Schmohl K.A., Schwenk N., Yen H.Y., Zach C., Schilling F., Weber W.A., Kälin R.E., Glass R., Nelson P.J., Wagner E, & Spitzweg C. (2021). Selective sodium iodide symporter (NIS) genetherapy of glioblastoma mediatedby EGFR-targeted lipopolyplexes. Molecular Therapy Oncolytics, 23, 432-446.
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2

Integrin Expression in Murine Bone Tissue

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After the last PET scan, mice were sacrificed, extremities fixed in 4 % PBS buffered formalin for 48 h, decalcified in Osteosoft (Merck, Darmstadt, Germany) for 4 weeks according to the manufacturer’s instructions, and embedded in paraffin. Sagittal sections of 4-μm thickness were cut, and consecutive sections stained with Movat pentachrome staining was performed using a staining kit (Morphisto, Frankfurt am Main, Germany). For immunohistochemistry (IHC), serial sections were stained using a Bond RXm system (Leica, Wetzlar, Germany, all reagents from Leica, unless otherwise indicated) with primary antibodies against α5-integrin (abcam, ab 108327, diluted 1:10.000) or β3-integrin (Cell Signaling, 13166S, diluted 1:300). Briefly, slides were deparaffinized using deparaffinization solution, pretreated with Epitope retrieval solution 2 (corresponding to EDTA buffer pH 8) for 30 min. Antibody binding was detected with a polymer refine detection kit without post-primary reagent and visualized with DAB as a dark brown precipitate. Slides were scanned (Leica AT2, Leica, Wetzlar, Germany) and evaluated using Imagescope Software (Leica, Wetzlar, Germany).
Notni J., Gassert F.T., Steiger K., Sommer P., Weichert W., Rummeny E.J., Schwaiger M., Kessler H., Meier R, & Kimm M.A. (2019). In vivo imaging of early stages of rheumatoid arthritis by α5β1-integrin-targeted positron emission tomography. EJNMMI Research, 9, 87.
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3

Histological Analysis of Tumor Specimens

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Histological analysis of tumor specimens was performed in five tumors per group. Tissues were fixed in 10% neutral-buffered formalin solution for minimally 48 h, dehydrated under standard conditions (Leica ASP300S, Wetzlar, Germany) and embedded in paraffin. Serial 2 μm sections were prepared with a rotary microtome (HM355S, ThermoFisher Scientific, Waltham, USA) and subjected to histological and immunohistochemical analysis. Hematoxylin-Eosin (H.-E.) staining was performed on deparaffinized sections with Eosin and Mayer’s Haemalaun according to a standard protocol. Immunohistochemistry was performed using a Bond RXm system (Leica, Wetzlar, Germany, all reagents from Leica) with a primary antibody against cleaved caspase 3 (clone ASP175, 1:150, Cell Signaling 9664). All slides were scanned with a high-throughput scanning system (AT2, Leica). The percentage of tumor area occupied by cleaved caspase 3 positive tumor cells and the average number of mitoses per 10 high power fields (MI) in tumor cells was evaluated in all sections by experienced pathologists (TG/KS), and representative images were taken using Aperio ImageScope × 64 (version 12.4.0.7018, Leica).
Schmidt O., Nehls N., Prexler C., von Heyking K., Groll T., Pardon K., Garcia H.D., Hensel T., Gürgen D., Henssen A.G., Eggert A., Steiger K., Burdach S, & Richter G.H. (2021). Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis. Journal of Experimental & Clinical Cancer Research : CR, 40, 322.
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4

Automated Immunohistochemistry Staining

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Immunohistochemistry (IHC) was performed with an automated immunostainer (Ventana Medical Systems) or Bond RXm system (Leica) according to the manufacturer’s protocols with minor modifications. Immunostaining was performed with the antibodies indicated in Table S4.
Mohr H., Ballke S., Bechmann N., Gulde S., Malekzadeh-Najafabadi J., Peitzsch M., Ntziachristos V., Steiger K., Wiedemann T, & Pellegata N.S. (2021). Mutation of the Cell Cycle Regulator p27kip1 Drives Pseudohypoxic Pheochromocytoma Development. Cancers, 13(1), 126.
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5

Immunohistochemistry on Human and Murine Tissues

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Immunohistochemistry on human TMAs and whole slide specimen as well as on murine tissues was performed using a Bond RXm system (Leica, Wetzlar, Germany, all reagents from Leica) with primary antibodies diluted as mentioned in the supplemental methods. Briefly, slides were deparaffinized using deparaffinization solution and pretreated with Epitope retrieval solution. Antibody binding was detected with a polymer refine detection kit without post-primary reagent and visualized with DAB as a dark brown precipitate. Counterstaining was done with hematoxylin.
Schick M., Zhang L., Maurer S., Maurer H.C., Isaakaidis K., Schneider L., Patra U., Schunck K., Rohleder E., Hofstetter J., Baluapuri A., Scherger A.K., Slotta-Huspenina J., Hettler F., Weber J., Engleitner T., Maresch R., Slawska J., Lewis R., Istvanffy R., Habringer S., Steiger K., Baiker A., Oostendorp R.A., Miething C., Lenhof H.P., Bassermann F., Chapuy B., Wirth M., Wolf E., Rad R., Müller S, & Keller U. (2022). Genetic alterations of the SUMO isopeptidase SENP6 drive lymphomagenesis and genetic instability in diffuse large B-cell lymphoma. Nature Communications, 13, 281.
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6

In Situ Hybridization for SARS-CoV-2 Detection

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ISH was conducted using the RNAscope2.5 LS Reagent Red Kit, according to the manufacturer’s instructions, and the Leica BOND-RXm system. Deparaffinization and heat-induced epitope retrieval were performed with BOND Epitope Retrieval Solution 2 (ER2; pH 9.0) for 25 minutes at 95°C. Hybridization for SARS-CoV-2 RNA was carried out using the RNAscope 2.5 LS Probe V-nCoV2019-S and checked for quality against slides treated with the positive control probe Hs-UBC and negative control Probe DapB. 
Cross A.R., de Andrea C.E., Villalba-Esparza M., Landecho M.F., Cerundolo L., Weeratunga P., Etherington R.E., Denney L., Ogg G., Ho L.P., Roberts I.S., Hester J., Klenerman P., Melero I., Sansom S.N, & Issa F. (2023). Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury. JCI Insight, 8(2), e157837.
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7

Immunohistochemical Analysis of CD31 in FFPE Tissue

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The formalin-fixed, paraffin-embedded (FFPE) tissue was used to prepare consecutive 2 μ m sections using a microtome (Microm). The immunohistochemistry (IHC) for CD31 (vessel endothelium) was performed with a primary rabbit anti-CD31 antibody (Abcam ab28364, 1:50) processed and detected on a Bond RXm system (Leica) with a polymer detection kit (without post primary antibody). The tissue was deparaffinized, pretreated with hydrogen-peroxide, incubated with the primary antibody for 15 min at room temperature, and the detection was performed with an anti-rabbit HRP polymer and DAB. Counterstaining was performed with hematoxylin. The slides were then dehydrated and coverslipped. Positive staining occurred as a dark-brown precipitate.
Birindelli G., Drobnjakovic M., Morath V., Steiger K., D’Alessandria C., Gourni E., Afshar-Oromieh A., Weber W., Rominger A., Eiber M, & Shi K. (2021). Is Hypoxia a Factor Influencing PSMA-Directed Radioligand Therapy?—An In Silico Study on the Role of Chronic Hypoxia in Prostate Cancer. Cancers, 13(14), 3429.
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8

Quantifying Autophagy Markers in Lung Tumors

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Sections were deparaffinized, rehydrated and subjected to epitope retrieval stained with a rabbit anti-LC3B (microtubule-associated protein 1A/11B-light chain 3) antibody (1:1000; NB100-2220, Novus Biologicals), mouse anti-CCS antibody (1:100; 55561, Santa Cruz Biotechnology), and rabbit anti-p62 antibody (1:1000; PM04, MBL International) by peroxidase-based detection and counterstaining with hematoxylin using the Leica Bond Rxm system. Photographs were taken on a Leica DMI6000B inverted light and fluorescent microscope (20x for quantitation, 5x or 40x for representative images). LC3- and p62-positive staining was assessed in Image J. The color deconvolution macro was applied to images while tumors were circumscribed with the freehand selection tool on the DAB (3,3′-diaminobenzidine) staining (Color_2) generated window. Using the threshold function, the total area of the tumor in pixels was recorded utilizing the same parameters for each tumor image. Areas staining positive by these parameters were selected and recorded in pixel units. The positive-staining area of the tumor in pixels was divided by the total area of the tumor in pixels to determine the percentage positive-staining area. Statistical analysis of abnormal lung area, % positive LC3 or positive p62 staining was analyzed using an unpaired, two-tailed Student’s t-test in Prism 7 (GraphPad).
Tsang T., Gu X., Davis C.I., Posimo J.M., Miller Z.A, & Brady D.C. (2022). BRAFV600E-Driven Lung Adenocarcinoma Requires Copper to Sustain Autophagic Signaling and Processing. Molecular Cancer Research, 20(7), 1096-1107.
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9

Multiplex IHC Analysis of Torpedo-like Structures

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Multiplex IHC was used to analyze the co-localization of KIF14, Mieap, and EZR proteins in torpedo-like structures. Multiplex IHC was performed with a Bond RXm system (Leica, Hamburg, Germany) with antibodies against KIF14 (HPA038061, 1:500, Sigma, St. Louis, MO, USA; detected by Opal 520), Mieap/SPATA18 (HPA036854, 1:100, Sigma, St. Louis, MO, USA; Opal 620), EZR (HPA021616, 1:1000, Sigma, St. Louis, MO, USA; Opal 690), and MMP13 (MA5-14238, 1:25, Thermo Fisher Scientific, Waltham, MA, USA). Protein blocking was performed using 3% BSA-PBS (Sigma, St. Louis, MO, USA). TSA visualization was performed with the Opal 520, Opal 620, and Opal 690 (Opal seven-color IHC kit, Perkin Elmer, Waltham, MA, USA). Staining was finished with a DAPI counterstain and slides were enclosed in fluorescence mounting medium (Agilent, Santa-Clara, CA, USA). Slides were scanned using the Vectra 3.0 (PerkinElmer, Waltham, MA, USA). Tissue imaging was performed using inForm Advanced Image Analysis software (inForm 2.1.1 and 2.2.1; Perkin Elmer, Waltham, MA, USA).
Gerashchenko T.S., Zolotaryova S.Y., Kiselev A.M., Tashireva L.A., Novikov N.M., Krakhmal N.V., Cherdyntseva N.V., Zavyalova M.V., Perelmuter V.M, & Denisov E.V. (2020). The Activity of KIF14, Mieap, and EZR in a New Type of the Invasive Component, Torpedo-Like Structures, Predetermines the Metastatic Potential of Breast Cancer. Cancers, 12(7), 1909.
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10

Kidney Histopathology and Apoptosis Quantification

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Kidneys were fixed in 4% formalin in PBS and embedded in paraffin. Sections of 2 μm were cut. Renal lesions were scored on sections stained with Hematoxylin-Eosin and PAS according to standard protocols. Cleaved Caspase 3 immunohistochemistry was performed on a BondRxm system (Leica, Wetzlar, Germany). Briefly, the slides were deparaffinized, and heat-mediated retrieval (ER1) was applied for 20 min. A primary antibody against cleaved Caspase 3 was used (1:150, Cell Signaling, Danvers, cat. No. 9664), and binding was visualized using a Polymer Refine Detection Kit (Leica, Wetzlar, Germany) without postprimary antibody. All slides were scanned with an AT2 scanning system (Leica, Wetzlar, Germany).
Renal tubular injury was scored by the percentage of injured tubules with loss of brush border, cell lysis, and cast formation: 0, no damage; 1, <25%; 2, 25 to 50%; 3, 50 to 75%; 4, >75% (60 (link)). Cleaved Caspase 3 positive tubular epithelial cells were counted in 10 high power fields (40×) and averaged.
Salei N., Ji X., Pakalniškytė D., Kuentzel V., Rambichler S., Li N., Moser M., Steiger K., Buch T., Anders H.J, & Schraml B.U. (2021). Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure. Proceedings of the National Academy of Sciences of the United States of America, 118(39), e2022311118.
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