Ix71 inverted multicolor fluorescent microscope

Manufactured by Olympus

The IX71 is an inverted multicolor fluorescent microscope designed for laboratory use. It is equipped with advanced optics and illumination systems to enable high-quality imaging of fluorescently labeled samples. The microscope's core function is to provide researchers with a versatile platform for visualizing and analyzing biological specimens.

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Lab products found in correlation

A21207, by Thermo Fisher Scientific (3 mentions) Dp71 camera, by Olympus (3 mentions) A21209, by Thermo Fisher Scientific (3 mentions) Ab6673, by Abcam (3 mentions) Donkey serum, by Merck Group (2 mentions) A11055, by Thermo Fisher Scientific (2 mentions) Rabbit anti β catenin, by Cell Signaling Technology (2 mentions) Goat anti gfp, by Abcam (2 mentions) Rat anti e cadherin, by Takara Bio (2 mentions) Aqua poly mount, by Polysciences (2 mentions) Af594 anti rabbit, by Thermo Fisher Scientific (1 mentions) Anti rat alexa594, by Thermo Fisher Scientific (1 mentions) Rat anti ki67, by Thermo Fisher Scientific (1 mentions) Anti p21, by Merck Group (1 mentions) Lsm 880 confocal microscope, by Zeiss (1 mentions) Rabbit anti nf kb p65, by Cell Signaling Technology (1 mentions) Rabbit anti phospho c jun, by Cell Signaling Technology (1 mentions) Rabbit anti prb ser807 811, by Cell Signaling Technology (1 mentions) Zrb1141, by Merck Group (1 mentions) Ab5690, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Inverted microscope (2417 citations) IX71 microscope (1097 citations) Fluorescent microscope (859 citations) IX71 inverted microscope (813 citations) Inverted fluorescent microscope (113 citations) IX71 fluorescent microscope (80 citations) IX71 inverted fluorescent microscope (38 citations)

9 protocols using ix71 inverted multicolor fluorescent microscope

Immunostaining protocol for protein analysis

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For immunostaining, tissues were fixed in Zn-formalin for 24 hr and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 min each, and then blocked in 5% donkey serum for 1 hr at room temperature, incubated with either Rabbit anti-BNIP3 antibody at 1:100 (A5683, Abclonal), Rabbit anti-NF-kB-p65 1:400 (8242S, Cell Signaling Technology), Rabbit anti-Phospho-c-jun 1:200 (3270S, Cell Signaling Technology), Rat anti-ki67 1:100 (14-5698-82, ebiosciences), Rabbit anti-pRB-Ser807/811 1:400 (8516, Cell Signaling Technology), anti-p21 1:1000 (ZRB1141, Sigma), Rat anti-Phospho H3 1:1000 (H9908, Sigma), or Goat anti-GFP (ab6673, Abcam) at 1:250 overnight at 4°C, washed, incubated with secondary antibody AF594-anti-rabbit (A-21207, Invitrogen), Alexa594 anti-rat (A-21209, Invitrogen), and Alexa488 anti-goat (11055, Invitrogen) antibodies at 1:200 for 1 hr at room temperature, counterstained with DAPI, washed, and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera using ×400 magnification. MFI was calculated by measuring average intensity over a fixed threshold for all images. NF-kB staining was imaged via Zeiss LSM880 confocal microscope using ×63 oil-immersion objective, and intensity was measured on 0.8 µm z-sections.

Sela Y., Li J., Kuri P., Merrell A.J., Li N., Lengner C., Rompolas P, & Stanger B.Z. (2021). Dissecting phenotypic transitions in metastatic disease via photoconversion-based isolation. eLife, 10, e63270.

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Immunohistochemical Analysis of Tumor-Infiltrating Cells

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For CD3 and Gr1 staining, collected implanted tumor tissues were fixed in Zn-formalin for 24 hours and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 minutes each, and then blocked in 5% donkey serum (Sigma, D9663) for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C, washed with 0.1% PBST (PBS with Tween-20), incubated with secondary antibodies for 1 hour at room temperature, and then washed and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera. For CD3 and Gr1 staining quantification, stained cells were counted for CD3+ T cells manually in 5–8 fields per sample.
Primary antibodies: CD3 (Abcam ab5690, 1:100 dilution) and Gr-1 (eBioscience 14-5931-85, 1:50 dilution), YFP (Abcam, ab6673). Secondary antibodies were purchased from Invitrogen (A-11055, A-21207, A-21209) and were used as 1:250 dilution for all staining.

Li J., Yuan S., Norgard R.J., Yan F., Sun Y.H., Kim I.K., Merrell A.J., Sela Y., Jiang Y., Bhanu N.V., Garcia B.A., Vonderheide R.H., Blanco A, & Stanger B.Z. (2020). Epigenetic and transcriptional control of the epidermal growth factor receptor (EGFR) regulates the tumor immune microenvironment in pancreatic cancer. Cancer discovery, 11(3), 736-753.

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Immunohistochemical Analysis of Mouse Pancreas

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Dissected mouse pancreata were fixed in zinc-formalin, processed, embedded in paraffin, sectioned and mounted. Immunohistochemical (IHC) sections were stained using a Bond Rx autostainer (Leica). Immunofluorescent sections were deparaffinized, rehydrated and antigen retrieval performed, which was followed by overnight incubation with the specific primary antibodies at 4°C. Primary antibodies used were PTHrP (Sigma AV33885), Ki-67 (Abcam ab16667), ECAD (Takara Clone M108), and GFP (Abcam; recognizes YFP). Images were acquired using an Olympus IX71 inverted multicolor fluorescent microscope equipped with a DP71 camera, and quantification performed in ImageJ software.

Pitarresi J.R., Norgard R.J., Chiarella A.M., Suzuki K., Bakir B., Sahu V., Li J., Zhao J., Marchand B., Wengyn M.D., Hsieh A., Kim I.K., Zhang A., Sellin K., Lee V., Takano S., Miyahara Y., Ohtsuka M., Maitra A., Notta F., Kremer R., Stanger B.Z, & Rustgi A.K. (2021). PTHrP drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability. Cancer discovery, 11(7), 1774-1791.

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Immunohistochemical Analysis of Tumor Markers

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For CD3, USP22, and Gr1 staining, collected implanted tumor tissues were fixed in Zn-formalin for 24 hours and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 minutes each, and then blocked in 5% donkey serum (Sigma, D9663) for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C, washed with 0.1% PBST (PBS with Tween-20), incubated with secondary antibodies for 1 hour at room temperature, and then washed and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera. For CD3 and Gr1 staining quantification, stained cells were counted for CD3⁺ T cells manually in 5-8 fields per sample.
Primary antibodies: CD3 (Abcam ab5690, 1:100 dilution), USP22 (Abcam ab195289, 1:100 dilution), Gr-1 (eBioscience 14-5931-85, 1:50 dilution), YFP (Abcam, ab6673). Secondary antibodies were purchased from Invitrogen (A-11055, A-21207, A-21209) and were used as 1:250 dilution for all staining.

Li J., Yuan S., Norgard R.J., Yan F., Yamazoe T., Blanco A, & Stanger B.Z. (2019). Tumor cell–intrinsic USP22 suppresses antitumor immunity in pancreatic cancer. Cancer immunology research, 8(3), 282-291.

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Multicolor Immunofluorescence Staining of Tissues

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Tissues were fixed in Zn-formalin for 24 hours and embedded in paraffin for histological analysis and immunofluorescence staining. Sections were deparaffinized, rehydrated and prepared by antigen retrieval for 5 minutes each, and then blocked in 5% donkey serum for 1 hour at room temperature, incubated with primary antibodies for overnight at 4°C, washed, incubated with secondary antibodies for 1 hour at room temperature, washed and mounted. Primary antibodies used include goat anti-GFP (Abcam), rabbit anti-CD3 (Abcam), rat anti-EMCN (Santa Cruz), rabbit anti-β-catenin (Cell signaling), rabbit anti-Collagen type I (Abcam), rabbit anti-PDGF Receptor β (Abcam), rabbit anti-alpha-SMA (Abcam), goat anti-SPARC (R&D). Slides were imaged and visualized using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera.

Li J., Byrne K.T., Yan F., Yamazoe T., Chen Z., Baslan T., Richman L.P., Lin J., Sun Y.H., Rech A.J., Balli D., Hay C.A., Sela Y., Merrell A.J., Liudahl S.M., Gordon N., Norgard R.J., Yuan S., Yu S., Chao T., Ye S., Eisinger-Mathason T.S., Faryabi R.B., Tobias J.W., Lowe S., Coussens L.M., Wherry E.J., Vonderheide R.H, & Stanger B.Z. (2018). Tumor cell-intrinsic factors underlie heterogeneity of immune cell infiltration and response to immunotherapy. Immunity, 49(1), 178-193.e7.

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In Vitro Cellular Cluster Generation

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In vitro clusters were generated as previous described^{17 (link)}. Briefly, adherent cells (PD7591 and PD798) were trypsinized and cultured in a 6 cm suspension culture dish with rocking for 12–16 h in a 37 °C incubator at 5% CO₂ at a density of approximately 1 million cells per ml in 5 ml media. 100 μl of cell suspension was then spiked into 0.5 ml of mouse blood and subjected to the sample preparation workflow described above. 1000 cells (events) were sorted into 4 wells of a 12-well plate for each sample. 5–6 individual images were collected per well and the counts were added to obtain a representative count for each well. The mean for 4 wells was reported. In order to assess the distribution of clusters prior to sorting, 1 ml of the cell suspension prior to spiking into blood was imaged in a 6 cm dish. 7 representative images were collected and size and number of clusters were quantified. All wells were stained with Hoechst 33342 and imaged on an Olympus IX71 inverted multicolor fluorescent microscope.

Bhagwat N., Dulmage K., Pletcher CH J.r., Wang L., DeMuth W., Sen M., Balli D., Yee S.S., Sa S., Tong F., Yu L., Moore J.S., Stanger B.Z., Dixon E.P, & Carpenter E.L. (2018). An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters. Scientific Reports, 8, 5035.

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Immunofluorescence staining of cells and tissues

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Tissues were fixed in Zn-formalin and embedded in paraffin prior to staining. Sections were deparaffinized, rehydrated and subjected to antigen retrieval. For staining cell lines, cells were fixed in 4% paraformaldehyde for 15 mins. For staining, sections and fixed cells were blocked in 5% donkey serum for 1 hour at room temperature (RT), incubated with primary antibodies for 1 hour at RT, washed, incubated with secondary antibodies for 1 hour at RT, washed and mounted. Primary antibodies used include goat anti-GFP (Abcam), rat anti-Ecadherin (Takara Bio), rabbit anti-Zeb1 (Santa Cruz), rabbit anti-Slug (gift of Dr. Joel Habener), rabbit anti-Vimentin (Cell Signaling Technologies), rabbit anti-Fsp1 (DAKO), rabbit anti-Rab5 (Cell Signaling Technologies), rabbit anti-Rab7 (Cell Signaling Technologies), rabbit anti-Rab11 (Cell Signaling Technologies), rabbit anti-EpCAM (Abcam), rabbit anti-Claudin-7 (Abcam), and rabbit anti-B-catenin (Cell Signaling Technologies). Zeb1 required additional tyramide signaling amplification (PerkinElmer). Slides were visualized using an Olympus IX71 inverted multicolor fluorescent microscope equipped with a DP71 camera. Select slides were also visualized using a Zeiss LSM 710 confocal microscope with Zen 2011 software.

Aiello N.M., Maddipati R., Norgard R.J., Balli D., Li J., Yuan S., Yamazoe T., Black T., Sahmoud A., Furth E.E., Bar-Sagi D, & Stanger B.Z. (2018). EMT subtype influences epithelial plasticity and mode of cell migration. Developmental cell, 45(6), 681-695.e4.

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Histological and Immunofluorescence Analysis

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Tissues were fixed in Zinc-formalin and embedded in paraffin for histological analysis and immunofluorescence staining. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval. For cell lines, cells were seeded into 8-well Nunc Lab-Tek II chamber slides (Thermo Scientific) and fixed in 4% paraformaldehyde for 15 mins. For immunofluorescence staining, sections or fixed cells were blocked in PBS with 0.3% Triton-X and 5% donkey serum for 1 hour, stained with primary and secondary antibodies, and mounted with Aqua Polymount (Polysciences, Inc). Primary antibodies used include goat anti-GFP (Abcam, ab6673), rat anti-E-cadherin (Takara Bio, M108), and rabbit anti-H3K36me2 (Abcam, ab9049). Slides were visualized using an Olympus IX71 inverted multicolor fluorescent microscope equipped with a DP71 camera. Images were quantified using ImageJ software.
For H&E staining, sections were deparaffinized, rehydrated, stained with hematoxylin, differentiated with acidic ethanol, stained for eosin, dehydrated, and mounted with Permount. Slides were visualized using the Keyence BZ-X710 all-in-one fluorescence microscope.

Yuan S., Natesan R., Sanchez-Rivera F.J., Li J., Bhanu N.V., Yamazoe T., Lin J.H., Merrell A.J., Sela Y., Thomas S.K., Jiang Y., Plesset J.B., Miller E.M., Shi J., Garcia B.A., Lowe S.W., Asangani I.A, & Stanger B.Z. (2020). Global regulation of the histone mark H3K36me2 underlies epithelial plasticity and metastatic progression. Cancer discovery, 10(6), 854-871.

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Histological and Immunofluorescence Analysis of Tissue Samples

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Tissues were fixed in Zinc-formalin and embedded in paraffin for histological analysis and immunofluorescence (IF) staining. For IF staining, sections were deparaffinized, rehydrated, and prepared by antigen retrieval. They were then blocked in PBS with 0.3% Triton-X and 5% donkey serum for 1 hour, stained with primary and secondary antibodies, and mounted with Aqua Polymount (Polysciences). Primary antibodies used include chicken anti-GFP (Abcam, ab13970) and rabbit anti-cleaved caspase-3 (Cell Signaling Technology, 9661). Slides were visualized using an Olympus IX71 inverted multicolor fluorescent microscope equipped with a DP71 camera. ImageJ FIJI software was used for quantification, with each data point an average of 2–3 fields per tumor section.
For H&E staining, sections were deparaffinized, rehydrated, stained with hematoxylin, differentiated with acidic ethanol, stained for eosin, dehydrated, and mounted with Permount. Slides were visualized using an Olympus BX41 microscope equipped with a DP25 camera.

Kim I.K., Diamond M., Yuan S., Kemp S., Li Q., Lin J., Li J., Norgard R., Thomas S., Merolle M., Katsuda T., Tobias J., Politi K., Vonderheide R, & Stanger B. (2023). Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy. Research Square.

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