Human BALF cell cytospin slides were fixed in 4% paraformaldehyde, incubated in blocking buffer (Aurion Blocking Solution, Electron Microscopy Sciences, Hatfield, PA) and reacted overnight with mouse anti-human CD68 (1:50 dilution, R & D Systems, Minneapolis, MN) and rabbit anti-human CD163 antibodies (1:50 dilution, Enzo Life Sciences, Inc., NY) diluted in 0.1% Aurion BSA-c (Electron Microscopy Sciences). Slides were washed 5 times in PBS, incubated with species-specific secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (Life Technologies, NY) at a 1:200 dilution, washed 5 times, mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and visualized utilizing a Zeiss LSM 510 UV confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).
Alexa fluor 488 or alexa fluor 568
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 and Alexa Fluor 568 are fluorescent dyes commonly used in biological research applications. Alexa Fluor 488 is a green-fluorescent dye, while Alexa Fluor 568 is an orange-fluorescent dye. These dyes can be used to label proteins, nucleic acids, and other biomolecules for detection and visualization in various experimental techniques, such as flow cytometry, microscopy, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 uv confocal microscope, by Zeiss (2 mentions)
Mouse anti human cd68, by R&D Systems (2 mentions)
Rabbit anti human cd163 antibodies, by Enzo Life Sciences (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
3 protocols using alexa fluor 488 or alexa fluor 568
1
Phenotyping of Human BALF Macrophages
2
Phenotyping of Human BALF Macrophages
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Human BALF cell cytospin slides were fixed in 4% paraformaldehyde, incubated in blocking buffer (Aurion Blocking Solution, Electron Microscopy Sciences, Hatfield, PA) and reacted overnight with mouse anti-human CD68 (1:50 dilution, R & D Systems, Minneapolis, MN) and rabbit anti-human CD163 antibodies (1:50 dilution, Enzo Life Sciences, Inc., NY) diluted in 0.1% Aurion BSA-c (Electron Microscopy Sciences). Slides were washed 5 times in PBS, incubated with species-specific secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 568 (Life Technologies, NY) at a 1:200 dilution, washed 5 times, mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and visualized utilizing a Zeiss LSM 510 UV confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).
3
EphB4 Receptor Antibody Characterization
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Cell lines used in this study were purchased from the American Type Culture Collection (ATCC) (Rockville, MD) and were cultured according to the ATCC recommendations in media and additives purchased from Life Technologies (Victoria, Australia). Three commercially available human EphB4-specific polyclonal antibodies EphB4 (N-19), EphB4 (H-200) and EphB4 (C-16) were purchased from Santa Cruz Biotechnology (Dallas, TX). An additional rabbit polyclonal antibody (S) recognizing human EphB4 (C-terminal) was a gift from Dr Andrew Ziemiecki, University of Bern, Switzerland [52 (link)]. For immunoprecipitation and Western blot analysis the C-terminal EphB4-specific mouse monoclonal antibody (Zymed, Life Technologies) and the phosphotyrosine specific 4G10 antibody (EMD Millipore, Billerica, MA) were used. The anti-human actin murine monoclonal antibody was from Abcam (Cambridge, MA). Secondary antibodies included a peroxidase-conjugated goat anti-mouse secondary antibody (Pierce, Thermo Fisher Scientific, Waltham, MA), a donkey anti-rabbit secondary antibody (Pierce) and Alexa Fluor® 488 or Alexa Fluor® 568, goat anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L) antibodies (Life Technologies).
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