Cell & Tissue Microscopy

 

Immunology

Multicolor fluorescence immunohistochemistry staining of mouse spleen tissue
Multicolor fluorescence immunohistochemistry staining of mouse spleen tissue
Spleen cryosections (5 µm) from mice expressing CX3CR1-green fluorescent protein (pseudo-colored green) and CCR2-red fluorescent protein (pseudo-colored red) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), blocked with 5% Goat serum and 1% BSA diluted in 1x PBS, and stained with BD Horizon™ BV421 Rat Anti-Mouse CD4 (Cat. No. 562891) (pseudo-colored magenta), Alexa Fluor® 647 Rat Anti-Mouse CD45R/B220 (Cat No. 557683) (pseudo-colored yellow), and BD Horizon™ BV480 Rat Anti-Mouse CD3 Molecular Complex (pseudo-colored cyan). Slides were mounted with ProLong® Gold and the image was captured on a BD Pathway™ 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision™ Software. 20X objective. Done in collaboration with the Catherine Hedrick lab at La Jolla Institute of Allergy and Immunology.
Multicolor fluorescence immunohistochemistry staining of mouse spleen tissue
Multicolor fluorescence immunohistochemistry staining of mouse spleen tissue
Spleen cryosections (5 µm) from wild-type mice were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), blocked with 5% Goat serum and 1% BSA diluted in 1x PBS, and stained with BD Horizon™ BV421 Rat Anti-Mouse F4/80 (Cat. No. 565411) (pseudo-colored red), BD Horizon™ BV480 Rat Anti-Mouse CD3 Molecular Complex (Cat. No. 565642) (pseudo-colored cyan), Alexa Fluor® 488 Rat Anti-Mouse B220 (Cat. No. 557669) (pseudo-colored green), Alexa Fluor® 555 Mouse Anti-Human Ki-67 (Cat. No. 558617) (pseudo-colored Yellow). Slides were nuclear counterstained with DRAQ5™ (Cat. No. 564902) (pseudo-colored blue) and mounted with ProLong® Gold. The image was captured on a BD Pathway™ 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision™ Software. 20X objective.

 

Cancer

Multicolor immunocytochemistry using BD Horizon™ BV480
Multicolor immunocytochemistry using BD Horizon™ BV480
MCF-7 cells (ATCC, HTB-22) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 and blocked with 5% goat serum, 1% BSA, 0.5% Triton™ X-100 in 1X PBS. Cells were incubated with Purified Mouse Anti-E-Cadherin (Cat. No. 610182) and the second step reagent was BD Horizon™ BV480 Goat Anti-Mouse (Cat. No. 564877) (pseudo-colored aqua). Cells were then stained with directly conjugated antibodies: Alexa Fluor® 488 Mouse anti-β-Tubulin (Cat No. 558605) (pseudo-colored green), Alexa Fluor® 555 Mouse anti-Human Ki-67 (Cat. No. 558617) (pseudo-colored purple), Alexa Fluor® 647 Mouse anti-GM130 (Cat. No. 558712) (pseudo-colored red). DAPI (Cat. No. 564907) was used as a nuclear counter-stain (pseudo-colored blue). Slides were mounted with ProLong® Gold antifade (Life Technologies) and the image was captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software (BD Biosciences). 20X objective.
Multicolor immunofluorescence staining of human U-2 OS cells
Multicolor immunofluorescence staining of human U-2 OS cells
Cultured cells from the human U-2 OS (Osteosarcoma, ATCC HTB-96) cell line were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), blocked with 5% Goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1x PBS, and stained with Purified Mouse Cytochrome C antibody (Cat. No. 556432) at 2.5 μg/mL. After washing, the cells were stained with the second step reagent, Goat BD Horizon™ BV480 Anti-Mouse Ig (Cat. No. 564877) (pseudo-colored green) at 2.5 μg/mL. After washing , the cells were then stained with BD Horizon™ BV421 Mouse Anti-Human CD107A (Cat. No. 562623) (pseudo-colored red), Alexa Fluor® 488 Rat Anti-Histone H3 (Cat. No. 558610) (pseudo-colored yellow), and Alexa Fluor® 555 Mouse Anti-Human Ki-67 (Cat No. 558617) (pseudo-colored cyan). DRAQ5 (Cat. No. 564902) was used as a nuclear counterstain (pseudo-colored blue). Slides were mounted with ProLong® Gold and the images were captured on a BD Pathway™ 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision™ Software. 40X objective.
Multicolor immunofluorescence staining of human Hela cells
Multicolor immunofluorescence staining of human Hela cells
Cultured cells from the human Hela (Adenocarcinoma, ATCC CCL-2) cell line were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), blocked with 5% Goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1X PBS, and stained with BD HorizonTM BV421 Mouse Anti-Human Lamp1 (Cat. No. 562623) (pseudo-colored green), unlabeled Mouse Anti-Cleaved PARP (Cat. No. 552597) with BD Horizon™ BV480 Goat Anti-Mouse second step (Cat. No. 564877) (pseudo-colored yellow), Alexa Fluor 488 Mouse Anti-Cytochrome C (Cat. No. 560263) (pseudo-colored red), and Alexa Fluor® 555 Mouse Anti-Human Ki-67 (Cat No. 558617) (pseudo-colored cyan). DRAQ5 (Cat. No. 564902) was used as a nuclear counterstain (pseudo-colored blue). Slides were mounted with ProLong® Gold and the images were captured on a BD Pathway™ 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision™ Software. 20X objective.

 

Stem Cell

Multicolor immunofluorescence staining of human embryonic stem cells
Multicolor immunofluorescence staining of human embryonic stem cells
Cultured cells from the H9 human ES cell line were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100 diluted in 1X PBS, blocked with 5% Goat serum, 1% BSA, and 0.5% Triton™ X-100 diluted in 1X PBS, and stained with BD Horizon™ BV421 Mouse Anti-SSEA-1 (Cat. No. 562705), BD Horizon BV480 Mouse Anti-CD324(E-Cadherin) (Cat. No. 565646), Alexa Fluor® 488 Mouse anti-Oct3/4 (Cat. No. 561628), and Alexa Fluor® 555 Mouse Anti-Human Ki-67 (Cat No. 558617). DRAQ5 (Cat. No. 564902) was used as a nuclear counterstain. Slides were mounted with ProLong® Gold and the images were captured on a BD Pathway™ 435 Cell Analyzer (epifluorescence microscope) and merged using BD Attovision™ Software. 20X objective.
Compatibility of BD Horizon BV480 with BD Horizon™ BV421 for immunofluorescence imaging
Compatibility of BD Horizon™ BV480 with BD Horizon™ BV421 for immunofluorescence imaging
Cells: H9 human embryonic stem cells (WiCell, Madison WI). Fix: BD Cytofix™ Fixation Buffer (Cat. No. 554655); Perm: BD Phosflow™ Perm Buffer III (Cat. No. 558050), Block/Stain: 5% goat serum, 1% BSA, 0.5 % Triton™ X-100 in 1X PBS. Antibodies: Purified Mouse anti-Oct3/4a (Cat. No. 561555) with BD Horizon™ BV480 Goat Anti-Mouse (Cat. No. 564877), BD Horizon™ BV421 Mouse Anti-Human TRA-1-60 Antigen (Cat. No. 562711). Other reagents: DRAQ5™ (BioStatus), ProLong® Gold antifade (Life Technologies). Instrument: BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software (BD Biosciences). 20X objective.

 

Fluorophore Characteristics

Spectral analysis
Spectral analysis

BD Horizon BV480 spillover into DAPI and Alexa Fluor® 488 channels is minimal
BD Horizon™ BV480 spillover into DAPI and Alexa Fluor® 488 channels is minimal
Human breast cancer cell line MCF-7 cells (ATCC, HTB-22) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with 0.1% Triton™ X-100, and blocked with 5% goat serum, 1% BSA, 0.5% Triton™ X-100 in 1X PBS. Cells were stained with BD Horizon™ BV480 Mouse Anti-Human GM130. Slides were mounted with ProLong® Gold antifade (Life Technologies) and the image was captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software (BD Biosciences). 20X objective.
Photostability of BV dyes
Photostability of BV dyes
Continuous exposure to excitation light. Cells: MCF7 cells. Fixation: BD Cytofix™ Fixation Buffer (Cat. No. 554655) Antibodies: Purified Mouse anti-Human CD324 (Cat No. 562869) with BD Horizon™ BV480 Goat anti-Mouse (Cat. No.564877), BD Horizon™ BV421 Goat-anti-Mouse (Cat. No. 563846), or Alexa Fluor® 488 Goat anti-Mouse (Life Technologies) Other reagents: ProLong® Gold antifade (Life Technologies) Instrument: ImageXpress® Micro XLS (Molecular Devices). 20X objective.
Very bright immunohistochemistry using BD Horizon BV480
Very bright immunohistochemistry using BD Horizon™ BV480
Frozen human colon sections were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeablized with 0.25% Triton™ X-100, blocked in 5% goat serum, 1% BSA, 0.2% gelatin, 0.2% Triton X in PBS, and stained with Purified Mouse Anti-E-Cadherin (Cat. No. 610182). The second step reagent was BD Horizon™ BV480 Goat Anti-Mouse (Cat. No. 564877) or  Alexa Fluor® 488 Goat Anti-Mouse (Life Technologies) (both pseudo-colored green) and nuclei were counter-stained with DRAQ5™ (BioStatus) (pseudo-colored red). Slides were mounted with ProLong® Gold antifade (Life Technologies) and the image was captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software (BD Biosciences). 20X objective.
Simple filter configuration for standard epifluorescence microscopy with 5 channels
Channel Fluor Excitation Dichoric Emission
Blue BV421/DAPI 392/23 409 430/24
Aqua BV480 438/24 458 485/20
Green 488 fluor 485/20 506 537/26
Orange 568 fluor 543/22 562 593/40
Far Red 647 fluor/Draq5 628/40 660 692/40
Simple filter configuration for standard epifluorescence microscopy with 5 channels


For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Alexa Fluor® and ProLong® are registered trademarks of Life Technologies Corporation.

Triton is a trademark of Sigma-Aldrich.

DRAQ5 is a trademark of BioStatus Ltd.

ImageXpress is a registered trademark of Molecular Devices.