Autostainer 360

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Autostainer 360 is a fully automated slide staining system designed for high-throughput immunohistochemistry and in situ hybridization applications in clinical and research laboratories. The instrument can process up to 60 slides simultaneously and provides consistent, reproducible staining results.

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Lab products found in correlation

A0485, by Agilent Technologies (1 mentions) M7239, by Agilent Technologies (1 mentions) Epitope retrieval solution ph 9, by Leica camera (1 mentions) Ultravision large volume detection system hrp polymer kit, by Thermo Fisher Scientific (1 mentions) Anti mouse, by Biocare Medical (1 mentions) Rodent block, by Biocare Medical (1 mentions) Dual endogenous enzyme block, by Agilent Technologies (1 mentions) Runx2, by Merck Group (1 mentions) Sftpb, by Thermo Fisher Scientific (1 mentions) Ab3786, by Cell Signaling Technology (1 mentions) Lgals1, by Cell Signaling Technology (1 mentions) Runx1, by Cell Signaling Technology (1 mentions) Sftpc, by Merck Group (1 mentions) Aperio scanscope system, by Leica Biosystems (1 mentions) Ab76013, by Abcam (1 mentions) Ta 125 hdx, by Thermo Fisher Scientific (1 mentions) Rabbit anti hmga2, by Cell Signaling Technology (1 mentions) β catenin, by BD (1 mentions) Neutral buffered formalin, by Avantor (1 mentions) Gemini stainer, by Thermo Fisher Scientific (1 mentions)

13 protocols using autostainer 360

Immunohistochemical Staining for EGFR and HER2

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Immunohistochemical staining of 3 µm TMA sections with diagnostically approved anti-EGFR (Clone E30, monoclonal mouse, M7239, DAKO, Hamburg, Germany, 1:10) or anti-ERBB2/HER2 antibodies (c-erbB-2, polyclonal rabbit, A0485, DAKO, 1:300) was performed on an autostainer 360 (Thermo Fisher Scientific, Waltham, USA) as previously specified [53 (link)]. For modifications see Supplementary Information.

Rose M., Maurer A., Wirtz J., Bleilevens A., Waldmann T., Wenz M., Eyll M., Geelvink M., Gereitzig M., Rüchel N., Denecke B., Eltze E., Herrmann E., Toma M., Horst D., Grimm T., Denzinger S., Ecke T., Vögeli T.A., Knuechel R., Maurer J, & Gaisa N.T. (2020). EGFR activity addiction facilitates anti-ERBB based combination treatment of squamous bladder cancer. Oncogene, 39(44), 6856-6870.

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IHC Staining of FFPE Placenta Tissues

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Human formalin-fixed paraffin-embedded (FFPE) first trimester placenta tissues (n = 31, mean gestational week 8.01 ± 2.08, see Suppl. Table 1) were cut (5 μm) and mounted on Superfrost Plus slides. After deparaffinization, slides were subjected to antigen retrieval by boiling sections in Epitope Retrieval Solution pH 9.0 (Novocostra, Leica) for 7 min at 120 °C. Thereafter, sections were stained using a staining robot (Autostainer 360; Thermo Fisher Scientific) with primary antibodies as indicated in Suppl. Table 3 using the UltraVision Large Volume Detection System HRP Polymer Kit (Thermo Fisher Scientific) as previously described [3 (link), 44 ].
Immunohistochemical double staining was performed with the Multivision Polymer Detection system using mouse monoclonal anti-HLA-G and rabbit polyclonal anti-vWF antibodies using dilutions as indicated in Suppl. Table 3 according to the protocol previously described [45 (link)].

Forstner D., Maninger S., Nonn O., Guettler J., Moser G., Leitinger G., Pritz E., Strunk D., Schallmoser K., Marsche G., Heinemann A., Huppertz B, & Gauster M. (2019). Platelet-derived factors impair placental chorionic gonadotropin beta-subunit synthesis. Journal of Molecular Medicine (Berlin, Germany), 98(2), 193-207.

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Immunohistochemistry Protocol for Sarcoma Tumors

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All immunohistochemistry procedures were performed essentially as described in Sánchez-Rivera et al. (36 (link)). Mice were killed by carbon dioxide asphyxiation. Sarcoma tumors were fixed with 4% paraformaldehyde (PFA) overnight, transferred to 70% ethanol, and subsequently embedded in paraffin. Sections were cut at a thickness of four micrometers and stained with H&E for routine pathological examination. Immunohistochemistry was performed on a Thermo Autostainer 360 machine. Slides were antigen-retrieved using Thermo citrate buffer, pH 6.0 in the pretreatment module. Sections were treated with Biocare rodent block, primary antibody, and anti-Mouse (Biocare) or anti-rabbit (Vector Labs) HRP-polymer. The slides were developed with Thermo Ultra DAB and counterstained with hematoxylin in a Thermo Gemini stainer and coverslipped using the Thermo Consul coverslipper. Cleaved caspase 3 was detected using anticleaved caspase 3 (CST, #9661; 1:1,000). All pictures were obtained using a Nikon 80i microscope with a DS-U3 camera and NIS-elements software.

Sánchez-Rivera F.J., Ryan J., Soto-Feliciano Y.M., Clare Beytagh M., Xuan L., Feldser D.M., Hemann M.T., Zamudio J., Dimitrova N., Letai A, & Jacks T. (2021). Mitochondrial apoptotic priming is a key determinant of cell fate upon p53 restoration. Proceedings of the National Academy of Sciences of the United States of America, 118(23), e2019740118.

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Immunohistochemical Profiling of RTK Targets

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For all IHC assays, 4-μm FFPE sections on positively charged slides were used. FFPE sections were deparaffinized on a Prisma autostainer (Sakura, Torrance, CA) and then subjected to antigen retrieval at high heat (98°C) with high pH (9.0) Tris-EDTA buffer (Dako, Carpinteria, CA) using a Lab Vision PT Module (Thermo Fisher Scientific, Carlsbad, CA). Following antigen retrieval, sections were stained with a pan-RTK antibody cocktail consisting of rabbit monoclonal antibodies, all obtained from Cell Signaling (Danvers, MA), targeting Pan-Trk (C17F1, active against TrkA, TrkB, and TrkC, 1:25 dilution), ROS1 (D4D6, 1:500), and ALK (D5F3, 1:500). Staining was performed using the Autostainer 360 (Thermo Fisher Scientific). Slides were blocked for both endogenous peroxidase and nonspecific protein binding by using Dual Endogenous Enzyme Block (Dako) and UV blocking reagent (Thermo Fisher Scientific). All other staining was performed primarily with DAKO series reagents (Rabbit Envision+Dual Link System-HRP, Mayer’s Hematoxylin, Liquid Diaminobenzinide (DAB)+substrate chromogen system). Specimens were scored positive by the pathologist if the specimens exhibited any intensity of stain (weak to strong) in >5% of tumor cells. Specimens without any visible or faint stain, in tumor cells, were scored negative.

Murphy D.A., Ely H.A., Shoemaker R., Boomer A., Culver B.P., Hoskins I., Haimes J.D., Walters R.D., Fernandez D., Stahl J.A., Lee J., Kim K.M., Lamoureux J, & Christiansen J. (2016). Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing. Applied Immunohistochemistry & Molecular Morphology, 25(7), 513-523.

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Immunohistochemical Analysis of Lung Tumors

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Individual lung tumors were fixed overnight in zinc formalin and embedded in paraffin. Tissue sections were dewaxed using a Thermo Autostainer 360. All sections from the same tumor regions were serially sectioned. Slides were then stained using antibodies against Nkx2.1 (1:1000), Hmga2 (1:1000), Onecut2, Proteintech, 21916-1-AP, 1:500 (in 1X PBST); Runx1, Cell Signaling Technology, 8529S, 1:500; Runx2, Cell signaling,12556S, 1:1000; Cav1, Sigma, C3237, 1:1000; Sftpb, ThermoFisher, PA5-42000, 1:200; Zeb1, Abcam, ab87280, 1:500; BATF, Sigma, SAB4500122, 1:100; Zfp95, Novus Biologicals, NBP2-20947, 1:200; RFP, Rockland, 600-401-379, 1:400; Fra1, ThermoFisher, PA5-40361, 1:100; CD45, Abcam, ab10558, 1:1000, Cell signaling technology 12556S, 1:1500; Sftpc, Millipore sigma AB3786, 1:5000; LGALS1, Cell Signaling Technology, 1388S, 1:1000. Slides were also counterstained with haematoxylin.

LaFave L.M., Kartha V.K., Ma S., Meli K., Priore I.D., Lareau C., Naranjo S., Westcott P., Duarte F.M., Sankar V., Chiang Z., Brack A., Law T., Hauck H., Okimoto A., Regev A., Buenrostro J.D, & Jacks T. (2020). Epigenomic state transitions characterize tumor progression in mouse lung adenocarcinoma. Cancer cell, 38(2), 212-228.e13.

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Lung Tissue Analysis of COVID-19 Patients

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With permission from patients’ families, lung samples from two patients who died of COVID19 and two control patients were collected in E.O. Mukhin Municipal Clinical Hospital (Moscow, Russia). The study was approved by the local Medical Ethical Committee (protocol #23/2020) and carried out following the rules of the Declaration of Helsinki of 1975. The diagnoses were confirmed by the history of symptoms, chest computed tomography scans, as well as real-time polymerase chain reaction. Tissue samples were fixed in 10% formalin, embedded into paraffin blocks and sectioned to 5 μm thickness. Immunohistochemistry (IHC-P) was performed as previously described [48 (link)]. Briefly, tissues were deparaffinized, and hydrated through an ethanol series. After antigen retrieval in citrate buffer pH 6 slides were processed in Autostainer 360 (Thermo Fisher, Waltham, MA, USA). Staining was visualized with DAB peroxidase substrate.

Antipova N.V., Larionova T.D., Siniavin A.E., Nikiforova M.A., Gushchin V.A., Babichenko I.I., Volkov A.V., Shakhparonov M.I, & Pavlyukov M.S. (2020). Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2. International Journal of Molecular Sciences, 21(23), 9167.

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Histopathologic Changes Quantification

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Histopathologic changes by FA were assessed at T5 and PT10. Tissue block preparation and histopathologic stains were conducted as reported previously [6 (link)] with minor modifications described below. For detection of involucrin, the antigen was retrieved in 1 mg/mL trypsin for 5 min at 37 °C. The immunohistochemical staining procedures after antigen retrieval were performed on an Autostainer 360 (ThermoFisher Scientific, Waltham, MA, USA) by going through the same steps as employed by manual staining. Stained sections were scanned and digital images obtained with the Aperio Scanscope System (Leica Biosystems, Vista, CA, USA). The percentages of Ki-67- or p63-positive nuclei were evaluated with the nuclear algorithm (Aperio Scanscope System). This algorithm counts the numbers of positive (brown) and negative (blue) nuclei present in the tissue section. The percentages of cleaved caspase-3-positive apoptotic bodies or periodic acid–Schiff (PAS)-stained goblet cells were evaluated semi-automatically, i.e., the total numbers of nuclei were counted with the nuclear algorithm, while the apoptotic bodies or PAS-stained goblet cells were counted manually.

Ren B., Wu Q., Muskhelishvili L., Davis K., Wang Y., Rua D, & Cao X. (2022). Evaluating the Sub-Acute Toxicity of Formaldehyde Fumes in an In Vitro Human Airway Epithelial Tissue Model. International Journal of Molecular Sciences, 23(5), 2593.

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Histological Analysis of Murine Tissues

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Mice were euthanized by CO2 asphyxiation and tissue was dissected, fixed in 10% neutral-buffered formalin overnight, and dehydrated in 70% ethanol prior to paraffin embedding. Adjacent 5-μm sections were cut and stained with hematoxylin and eosin or picosirius red or subject to immunohistochemistry (IHC). Primary antibodies for IHC are listed in the Key Resource Table. Mach 2 HRP-labeled micro-polymers (Biocare Medical) were used for primary antibody detection using a Thermo Scientific Autostainer 360. Slides were imaged with a modified Nikon T2R inverted microscope (MVI), 4x/10x/20x/40x objectives, and a 2.8 MP CoolSNAP Dyno CCD camera (Photometrics). Monochromatic red, green, and blue images were merged using ImageJ software (NIH).

Chung K.M., Singh J., Lawres L., Dorans K.J., Garcia C., Burkhardt D.B., Robbins R., Bhutkar A., Cardone R., Zhao X., Babic A., Vayrynen S.A., Costa A.D., Nowak J.A., Chang D.T., Dunne R.F., Hezel A.F., Koong A.C., Wilhelm J.J., Bellin M.D., Nylander V., Gloyn A.L., McCarthy M.I., Kibbey R.G., Krishnaswamy S., Wolpin B.M., Jacks T., Fuchs C.S, & Muzumdar M.D. (2020). Endocrine-exocrine signaling drives obesity-associated pancreatic ductal adenocarcinoma. Cell, 181(4), 832-847.e18.

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Immunohistochemical Profiling of Lung Tissues

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Sectioned organoids or tissues were stained with hematoxylin and eosin (H&E) or IHC-stained with the following antibodies: for organoids, anti-TTF1 (1:500; Abcam ab76013), rabbit anti-SFTPC (1:5000; Millipore ABC99), rabbit anti-Caveolin-1 (1:5000; Thermo C3237), rabbit anti-CCSP (1:5000; Millipore 07-623), and rabbit anti-Keratin 5 (1:5000; Biolegend 905501); for tissues, rabbit anti-TTF1 (1:500; Abcam ab76013) and rabbit anti-HMGA2 (1:400; Cell Signaling Technology 8179). For IHC staining of lung and organoid sections, antigen retrieval was performed in IHC retrieval solution (pH 6.0; Thermo TA250) for 20 min at 97°C. Slides were processed using a Thermo Scientific Autostainer 360 with the following run conditions: endogenous peroxidases (Thermo TA125H2O2q) blocking for 10 min, protein block (Biocare RBM96961L) for 30 min, primary antibody for 60 min, and labeled polymer (Biocare RMR622L) for 30 min with a 5-min DAB (Thermo TA125HDX) exposure.

Naranjo S., Cabana C.M., LaFave L.M., Romero R., Shanahan S.L., Bhutkar A., Westcott P.M., Schenkel J.M., Ghosh A., Liao L.Z., Del Priore I., Yang D, & Jacks T. (2022). Modeling diverse genetic subtypes of lung adenocarcinoma with a next-generation alveolar type 2 organoid platform. Genes & Development, 36(15-16), 936-949.

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Immunohistochemical Analysis of Tumor Markers

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Tissues or tumour organoids were fixed in 10% formalin overnight and embedded in paraffin. Immunohistochemistry (IHC) was performed on a Thermo Autostainer 360 with or without hematoxylin counterstaining using antibodies to β-catenin (BD, cat. # 610153), Ki67 (Vector Labs, cat. # VP-RM04), glutamine synthetase (BD, cat. # 610517), or Porcupine (AbCam, cat. # ab105543). Lungs from at least three tumor-bearing mice were analysed by each antibody. Livers and small intestines harvested from three normal, healthy mice were subjected to β-catenin, glutamine synthetase, and Porcupine IHC. 65 human LUAD tumors samples in two separate tissue microarrays were analysed by β-catenin and Porcupine IHC. 5 human colorectal adenocarcinoma samples were stained with Porcupine antibodies. All human tissue material was obtained commercially from Janssen Pharmaceuticals.

Tammela T., Sanchez-Rivera F.J., Cetinbas N.M., Wu K., Joshi N.S., Helenius K., Park Y., Azimi R., Kerper N.R., Wesselhoeft R.A., Gu X., Schmidt L., Cornwall-Brady M., Yilmaz Ö.H., Xue W., Katajisto P., Bhutkar A, & Jacks T. (2017). A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma. Nature, 545(7654), 355-359.

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