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Dm6000b epifluorescent microscope

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The Leica DM6000B is an epifluorescent microscope designed for advanced fluorescence imaging applications. It features a high-intensity mercury or xenon illumination system, a wide range of fluorescence filter cubes, and a motorized focusing mechanism. The DM6000B enables researchers to perform a variety of fluorescence-based analyses and experiments.

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Lab products found in correlation

Prolong antifade mounting medium, by Thermo Fisher Scientific (7 mentions) Tween 20, by Merck Group (3 mentions) Optimal cutting temperature compound, by Sakura Finetek (3 mentions) Fc block anti cd16 cd32 2.4g2, by Cytek Biosciences (2 mentions) Goat serum, by Jackson ImmunoResearch (2 mentions) Donkey anti goat af555, by Abcam (1 mentions) Cd45.1 clone a20, by BioLegend (1 mentions) Cd8α 53 6, by Cytek Biosciences (1 mentions) Cm1950 cryostat, by Leica (1 mentions) Goat anti rabbit af555, by Thermo Fisher Scientific (1 mentions) Ulex europaeus agglutinin 1 uea 1, by Vector Laboratories (1 mentions)

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DM6000B (555 citations) DM6000B microscope (357 citations) Epifluorescent microscope (13 citations) DM6000B epifluorescent upright microscope (11 citations)

9 protocols using dm6000b epifluorescent microscope

1

Quantification of Lung Cell Populations

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B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice previously infected with PR8 or IAV_Cre were euthanized at indicated dpi. Lungs were harvested and fixed in 2% PFA for two hours, immersed overnight in 30% sucrose, inflated and flash frozen in optimum cutting temperature compound (O.C.T.). 7 μm sections were cut from each block with a Leica CM 1860 cryostat and stained prior to imaging on a Leica DM6000B EPI fluorescent microscope (violet LED). Histo-cytometry was performed using Imaris (Bitplane) by creating surfaces for the reporter+ cells (tdTomato) and markers (SPC or CC10), and performing a distance transformation to detect double positive cells. Reporter+ cells were manually counted in ImageJ. Neighbor cells were defined as two or more reporter+ DAPI+ cells within the proximity of one DAPI-stained nucleus to one another. Abs used were: CC10 (polyclonal) (Abcam), proSP-C (polyclonal) (Millipore Sigma), goat anti-rabbit (polyclonal) (ThermoFisher Scientific).
Fiege J.K., Stone I.A., Dumm R.E., Waring B.M., Fife B.T., Agudo J., Brown B.D., Heaton N.S, & Langlois R.A. (2019). Long-term surviving influenza infected cells evade CD8+ T cell mediated clearance. PLoS Pathogens, 15(9), e1008077.
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2

Immunofluorescence Staining of Tissue Sections

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Thymi embedded in optimal cutting temperature compound (Sakura Finetek) were sectioned (7-μm slices) at −20 °C, followed by fixation and permeabilization in 100% acetone for 20 min at 4 °C. Acetone fixed sections were then blocked for 60 min at room temperature with 5% bovine serum albumin (BSA) and Fc block (clone 2.4G2). Following washing, sections were stained in 0.5% BSA and 0.1% Tween-20 (Sigma-Aldrich) overnight at 4 °C. Stained sections were washed, stained with DAPI, and mounted using Prolong antifade mounting medium (Life Technologies). Images of sections were acquired with a Leica DM6000B epifluorescent microscope
Martinez R.J., Breed E.R., Worota Y., Ashby K.M., Vobořil M., Mathes T., Salgado O.C., O’Connor C.H., Kotenko S.V, & Hogquist K.A. (2023). Type III interferon drives thymic B cell activation and regulatory T cell generation. Proceedings of the National Academy of Sciences of the United States of America, 120(9), e2220120120.
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3

Multimodal Immune Profiling of Thymic Tissue

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Thymi were harvested and snap frozen in Optimal Cutting Temperature compound (Sakura Finetek). Tissue samples were sectioned into 7μm at −20°C. Sections were then fixed and permeabilized in 100% acetone for 20 minutes at 4°C. Fixed sections were blocked with 5% bovine serum albumin (BSA) and Fc block (anti-CD16/CD32; 2.4G2, Tonbo Biosciences) for one hour at 20°C. Antibodies were purchased from BD Biosciences: CD8α (53–6.7), CD11c (HL3), BioLegened: CD301b (MGL2; URA-1), and Vector Laboratories: Fluorescein labeled Ulex Europaeus Agglutinin I (UEAI). Sections were stained with an antibody cocktail in 0.5% BSA and 0.1% Tween-20 (Sigma Aldrich) overnight at 4°C. Following wash steps and DAPI staining, sections were mounted using Prolong anti-fade mounting medium (Life Technologies). Images were acquired using a Leica DM6000B epifluorescent microscope 16–72 hours later.
Breed E.R., Vobořil M., Ashby K.M., Martinez R.J., Qian L., Wang H., Salgado O.C., O’Connor C.H, & Hogquist K.A. (2022). Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion. Nature immunology, 23(7), 1042-1051.
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4

Immunofluorescence Analysis of Thymus

Cited 2 times
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Immunofluorescence analysis of thymi was described previously 51 (link). Thymi were washed, fixed with 4% paraformaldehyde (PFA) for 1 h and snap frozen. 5-μm sections were blocked with PBS containing 5% bovine serum albumin (BSA) and goat serum (Jackson Laboratory) before staining. The sections were then covered with Prolong anti-fade mounting medium (Life Technologies) and images were obtained 1 to 3 d later with a Leica DM6000B Epi-Fluorescent microscope.
Histo-cytometry analysis was performed as described previously 21 (link).
Owen D.L., Mahmud S.A., Sjaastad L.E., Williams J.B., Spanier J.A., Simeonov D.R., Ruscher R., Huang W., Proekt I., Miller C.N., Hekim C., Jeschke J.C., Aggarwal P., Broeckel U., LaRue R.S., Henzler C.M., Alegre M.L., Anderson M.S., August A., Marson A., Zheng Y., Williams C.B, & Farrar M.A. (2019). Thymic regulatory T cells arise via two distinct developmental programs. Nature immunology, 20(2), 195-205.
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5

Immunohistochemical Analysis of Thymic Cell Populations

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Thymi were harvested and snap frozen in Optimal Cutting Temperature compound (Sakura Finetek). 7μm sections were fixed and permeabilized in 100% acetone for 20 minutes at 4° C. Samples were then blocked with 5% bovine serum albumin (BSA) and Fc block (anti-CD16/CD32; 2.4G2, Tonbo Biosciences) for one hour at 20°C prior to staining. Antibodies were purchased from BD Biosciences: CD11c (HL3), BioLegened: F4/80 (BM8), CD86 (GL-1), Tonbo biosciences: CD80 (16–10A1), and Vector Laboratories: Fluorescein labeled Ulex Europaeus Agglutinin I (UEA I). Blocked sections were stained with desired antibodies combined into a cocktail in 0.5% BSA and 0.1% Tween-20 (Sigma Aldrich) overnight at 4°C prior to DAPI staining. Sections were mounted using Prolong anti-fade mounting medium (Life Technologies). Images were acquired using a Leica DM6000B epifluorescent microscope 16–72 hours later.
Breed E.R., Watanabe M, & Hogquist K.A. (2019). Measuring thymic clonal deletion at the population level. Journal of immunology (Baltimore, Md. : 1950), 202(11), 3226-3233.
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6

Immunofluorescence Analysis of Thymus

Cited 2 times
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Immunofluorescence analysis of thymi was described previously 51 (link). Thymi were washed, fixed with 4% paraformaldehyde (PFA) for 1 h and snap frozen. 5-μm sections were blocked with PBS containing 5% bovine serum albumin (BSA) and goat serum (Jackson Laboratory) before staining. The sections were then covered with Prolong anti-fade mounting medium (Life Technologies) and images were obtained 1 to 3 d later with a Leica DM6000B Epi-Fluorescent microscope.
Histo-cytometry analysis was performed as described previously 21 (link).
Owen D.L., Mahmud S.A., Sjaastad L.E., Williams J.B., Spanier J.A., Simeonov D.R., Ruscher R., Huang W., Proekt I., Miller C.N., Hekim C., Jeschke J.C., Aggarwal P., Broeckel U., LaRue R.S., Henzler C.M., Alegre M.L., Anderson M.S., August A., Marson A., Zheng Y., Williams C.B, & Farrar M.A. (2019). Thymic regulatory T cells arise via two distinct developmental programs. Nature immunology, 20(2), 195-205.
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7

CD1d Tetramer Immunofluorescence Staining Protocol

Cited 1 time
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The CD1d tetramer immunofluorescence has been described previously (Lee et al., 2015 (link)). Briefly, fresh thymic lobes were incubated with PE-CD1d-PBS57 tetramer at 4°C overnight. The tissues were then washed with PBS and fixed in 4% paraformaldehyde (PFA) for 1 hr and snap frozen in OCT. Five micrometer sections were made and stained with Goat-anti-R Phycoerythrin (PE) (Abcam) followed by Donkey-anti-Goat AF555 (Abcam). The sections were incubated with Rabbit-anti-β5t (MBL International) and anti-CD45.1 AF647 (Biolegend) at 4°C overnight, followed by Goat-anti-Rabbit BV480 (BD biosciences). The sections were counterstained with DAPI covered with Prolong anti-fade mounting medium (Life Technologies). The images were obtained with a Leica DM6000B Epi-Fluorescent microscope.
Wang H, & Hogquist K.A. (2018). CCR7 defines a precursor for murine iNKT cells in thymus and periphery. eLife, 7, e34793.
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8

Quantifying lung-infiltrating CD8+ cells

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C57Bl/6 mice previously infected with IAV_N4 or IAV_T4 were injected iv with anti-CD8α for three minutes prior to takedown. Lungs were fixed in 2% PFA, inflated and flash frozen in optimum cutting temperature compound. 7 μm sections were cut from each block with a Leica CM1950 cryostat and stained for imaging on a Leica DM6000B EPI fluorescent microscope (violet LED). A minimum of 500 and a maximum of 30,000 CD45.1+/CD8α− cells per section were counted using ImageJ software for a percentage of the total number of nucleated cells. Abs used were: CD8α (53–6.7) (Tonbo), CD45.1 (clone A20) (Biolegend) and donkey anti-rat (polyclonal) (Jackson ImmunoResearch).
Fiege J.K., Stone I.A., Fay E.J., Markman M.W., Wijeyesinghe S., Macchietto M.G., Shen S., Masopust D, & Langlois R.A. (2019). THE IMPACT OF TCR SIGNAL STRENGTH ON RESIDENT MEMORY T CELL FORMATION DURING INFLUENZA VIRUS INFECTION. Journal of immunology (Baltimore, Md. : 1950), 203(4), 936-945.
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9

Immunohistochemical Analysis of Thymic Architecture

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Thymi were fixed with 4% paraformaldehyde overnight, transferred to 30% sucrose solution for 24 h, and snap frozen in optimum cutting temperature compound. Sections (7 μm) were blocked with 5% bovine serum albumin and Fc block (anti-CD16/CD32; 2.4G2, Tonbo Biosciences) in dilution 1 : 100 for 1 h at room temperature (RT) prior to staining. The sections were incubated with Rabbit-anti-b5t in dilution 1 : 200 (MBL International) and fluorescein-labeled Ulex europaeus agglutinin I (UEA-I) (Vector Laboratories) at 4 °C overnight, followed by Goat-anti-Rabbit-AF555 in dilution 1 : 500 for 1 h at RT (Thermo Fisher Scientific). Sections were next stained with 4′,6-diamidino-2-phenylindole and mounted using ProLong antifade mounting medium (Life Technologies). Images were acquired using a Leica DM6000B epifluorescent microscope.
Georgiev H., Peng C., Huggins M.A., Jameson S.C, & Hogquist K.A. (2021). Classical MHC expression by DP thymocytes impairs the selection of non-classical MHC restricted innate-like T cells. Nature Communications, 12, 2308.
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