Bond rxm autostainer

Manufactured by Leica

Sourced in Germany, United States

The Bond RXm autostainer is a fully automated system designed for immunohistochemistry (IHC) and in situ hybridization (ISH) staining of tissue samples. The system automates the staining process, including deparaffinization, antigen retrieval, primary antibody incubation, and detection, to provide consistent and reliable results.

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Lab products found in correlation

Opal 7 color automation ihc kit, by Akoya Biosciences (2 mentions) Opal automation ihc kit, by Akoya Biosciences (2 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Er2 solution, by Leica (1 mentions) Vectra polaris automated quantitative pathology imaging system, by Akoya Biosciences (1 mentions) Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions) Anti cd8 clone epr20305, by Abcam (1 mentions) Anti foxp3 clone 1054c, by Novus Biologicals (1 mentions) Anti pan cytokeratin rabbit poly, by Bioss Antibodies (1 mentions) Prolong diamond, by Thermo Fisher Scientific (1 mentions) Anti ki 67 clone d3b5, by Cell Signaling Technology (1 mentions) Anti dig clone 9h27l19, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Bond RXm (65 citations) Bond Autostainer (48 citations) Autostainer (38 citations)

7 protocols using bond rxm autostainer

In Situ Hybridization for Transcriptomic Analysis

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To prepare slides for DSP, 4-μm thick TMA sections were deparaffinized, heated in ER2 solution (Leica) at 100 °C for 20 min, and treated with 1 µg/ml proteinase K (Ambion) at 37 °C for 15 min on a BOND RXm autostainer (Leica). An overnight in situ hybridization was performed as described^{71 (link)} with a final probe concentration of 4 nM per probe. The panel included probes that target 2106 mRNA transcripts as well as 220 negative probes (18,120 probes total, median 10 probes per target). Slides were washed twice at 37 °C for 25 min with 50% formamide/2X SSC buffer to remove unbound probes.

Brady L., Kriner M., Coleman I., Morrissey C., Roudier M., True L.D., Gulati R., Plymate S.R., Zhou Z., Birditt B., Meredith R., Geiss G., Hoang M., Beechem J, & Nelson P.S. (2021). Inter- and intra-tumor heterogeneity of metastatic prostate cancer determined by digital spatial gene expression profiling. Nature Communications, 12, 1426.

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Immunohistochemical Profiling of Cellular Markers

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All IHC studies were performed on 5 µm FFPE sections using a Leica BOND RXm autostainer. Slides were stained with antibodies targeting human P-S6 (Cell Signaling #4858, 1:100) and P-PRAS40 (Cell Signaling #13175, 1:200) using a modified version of either the standard Leica Bond DAB “F” or red “J” IHC protocols. Slides were scored by a board-certified pathologist and given an H-score based on percentage and intensity of positivity. CD3, CD8, PAX5, and PTEN staining and analyses were performed as previously described.^{8 (link)}

Fischer G.M., Guerrieri R.A., Hu Q., Joon A.Y., Kumar S., Haydu L.E., McQuade J.L., Vashisht Gopal Y.N., Knighton B., Deng W., Hudgens C.W., Lazar A.J., Tetzlaff M.T, & Davies M.A. (2021). Clinical, molecular, metabolic, and immune features associated with oxidative phosphorylation in melanoma brain metastases. Neuro-oncology Advances, 3(1), vdaa177.

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Multiplex Immunohistochemistry of OT

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For culture, three fresh OT samples were either dissected into 5-mm explants or dissociated with mechanical disruption and brief trypsinization (0.5% trypsin at room temperature). For histology on three OT samples (Supplementary Table 4), commercial antibodies were validated on tonsil tissue with chromogenic and multiplex fluorescent approaches using the Leica BOND RXm autostainer (Supplementary Table 3 and Supplementary Figs 3 and 4). Then, serial 5-μm formaldehyde-fixed paraffin embedded (FFPE) OT sections, and a post-mortem lymph node from a patient with NMDAR-antibody encephalitis,^{28 (link)} were stained with the single chromogenic and three multiplex panels (all commercial antibodies listed in Supplementary Table 3). These were digitally scanned using the Vectra Polaris automated quantitative pathology imaging system (Akoya Biosciences) and images extracted using the HALO software package (Indica Laboratories).

Al-Diwani A., Theorell J., Damato V., Bull J., McGlashan N., Green E., Kienzler A.K., Harrison R., Hassanali T., Campo L., Browne M., Easton A., Soleymani majd H., Tenaka K., Iorio R., Dale R.C., Harrison P., Geddes J., Quested D., Sharp D., Lee S.T., Nauen D.W., Makuch M., Lennox B., Fowler D., Sheerin F., Waters P., Leite M.I., Handel A.E, & Irani S.R. (2022). Cervical lymph nodes and ovarian teratomas as germinal centres in NMDA receptor-antibody encephalitis. Brain, 145(8), 2742-2754.

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Multiplex IHC for Tumor-Infiltrating Tregs

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To evaluate the binding antibody to intratumoral Tregs, multiplex immunohistochemistry (IHC) for tumor infiltrating lymphocytes (TILs) in tissue sections was carried out using Opal 7-Color Automation IHC Kit (Akoya Biosciences, Hopkinton, MA) and BOND RXm auto stainer (Leica Biosystems, Wetzlar, Germany). Anti-CTLA4-IgG (1 mg, 6.8 nmol) or antiCTLA4-F(ab′)₂ (0.73 mg, 6.8 nmol) antibody was conjugated with a 5-fold molar excess of digoxigenin (DIG) NHS ester (MilliporeSigma) in Na₂HPO₄ (pH 8.5) at room temperature for 1 h. The mixture was purified with a Sephadex G25 column (PD-10). Each DIG-conjugated antibody was injected into MC38-luc tumor-bearing mouse via tail vein. The tumors were extracted 24 h after injection, then fixed with 10% formalin, embedded in paraffin, and thinly sliced. The following antibodies and DAPI were used: anti-CD8 (clone EPR20305; Abcam, 1:500 dilution), anti-Foxp3 (clone 1054C; Novus Biologicals, 1:1,000 dilution), and anti-DIG (clone 9H27L19; Thermo Fisher Scientific, 1:500 dilution) antibodies. The staining using this system was carried out as previously described.^{25 (link)} Stained slides were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were captured by Mantra Quantitative Pathology Workstation (AKOYA Biosystems) and were analyzed with inForm software (AKOYA Biosystems).

Auland M.E., Roufogalis B.D., Devaux P.F, & Zachowski A. (1994). Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.. Proceedings of the National Academy of Sciences of the United States of America, 91(23), 10938-10942.

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Multicolor Immunofluorescence Staining of Tumor-Infiltrating Lymphocytes

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Multicolor immunofluorescence staining was performed to analyze the tumor-infiltrating lymphocytes using Opal 7-color Automation IHC Kit (Akoya Bioscience, Menlo Park, CA, USA) and Bond RXm auto stainer (Leica Biosystems, Wetzlar, Germany) according to the protocol previously described [18 (link)]. Three pictures were taken for each specimen, and each parameter was summed. Cell density was calculated as cell counts per square millimeter.

Okada R., Kato T., Furusawa A., Inagaki F., Wakiyama H., Fujimura D., Okuyama S., Furumoto H., Fukushima H., Choyke P.L, & Kobayashi H. (2022). Selection of antibody and light exposure regimens alters therapeutic effects of EGFRtargeted near-infrared photoimmunotherapy. Cancer immunology, immunotherapy : CII, 71(8), 1877-1887.

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Multiplex IHC for Tissue Characterization

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Multiplex IHC was performed as previously described (34 (link)), using Opal Automation IHC Kit (Akoya Bioscience) and Bond RXm autostainer (Leica Biosystems). The following antibodies were used: anti-pan cytokeratin (rabbit poly; RRID: AB_10855057, Bioss), anti-PDPN (clone 66; RRID: AB_2785565, Invitrogen), anti-αSMA (clone EPR5368; RRID: AB_11129103, Abcam), anti-CD31 (clone D8V9E; RRID: AB_2722705, Cell Signaling Technology), anti-VEGFR3 (clone AFL4; RRID: AB_467795, Thermo Fisher Scientific), and anti-digoxigenin (DIG, clone 9H27L19; RRID: AB_2532342, Thermo Fisher Scientific; 1:500). Cells were counterstained with DAPI. Stained slides were imaged with Mantra Quantitative Pathology Workstation (Akoya Biosystems), and images were analyzed with inForm software (RRID: SCR_019155, Akoya Biosystems).

Kato T., Furusawa A., Okada R., Inagaki F., Wakiyama H., Furumoto H., Fukushima H., Okuyama S., Choyke P.L, & Kobayashi H. (2023). Near-Infrared Photoimmunotherapy Targeting Podoplanin-Expressing Cancer Cells and Cancer-Associated Fibroblasts. Molecular cancer therapeutics, 22(1), 75-88.

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Multiplex IHC for Tumor Cell Profiling

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Multiplex immunohistochemistry was carried out as described previously
¹⁸ (link) using an Opal Automation IHC Kit (Akoya Bioscience) and Bond RXm autostainer (Leica Biosystems). The sections were stained with DAPI and the following Abs: anti‐CD45 (clone D3F8Q; Cell Signaling Technology; 1:500), anti‐DIG (clone 9H27L19; Thermo Fisher Scientific; 1:500), anti‐Ki‐67 (clone D3B5; Cell Signaling Technology; 1:500), and anti‐pCK (rabbit poly; Bioss Antibodies; 1:250). Stained slides were mounted with ProLong Diamond (Thermo Fisher Scientific) and imaged with a Mantra Quantitative Pathology Workstation (Akoya Biosystems). The obtained images were analyzed with inForm Tissue Finder software (Akoya Biosystems). To calculate the percentage of Ki‐67 positive cancer cells, inForm software was trained to detect tissue and cell phenotypes using machine‐learning algorithms based on the following criteria: areas with pCK expression = tumor, other areas = stroma, pCK+ CD45− cells = cancer cells, pCK− CD45+ = blood cells, and pCK− CD45− = other cells. inForm software computed the percentage of Ki‐67‐positive cells among cancer cells. The average percentage was calculated from five images for each specimen.

Takao S., Fukushima H., King A.P., Kato T., Furusawa A., Okuyama S., Kano M., Choyke P.L., Escorcia F.E, & Kobayashi H. (2023). Near‐infrared photoimmunotherapy in the models of hepatocellular carcinomas using cetuximab‐IR700. Cancer Science, 114(12), 4654-4663.

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