Cell-permeable, yellow fluorescent dye supplied as an NHS ester / succinimidyl ester (SE) for coupling to primary amine (NHS) groups. NHS esters are used to label the primary amines of proteins and are commonly used for conjugating dyes to a protein or antibody.
May be used for cellular imaging when combined with the HaloTag or SNAP-tag self labelling systems. Suitable for confocal microscopy and super resolution microscopy (SRM) including techniques such as dSTORM (both live and fixed cells). Can also be multiplexed with Janelia Fluor ® 635 SE for two color imaging.
Fig 1. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody.
Figure 1. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody. Rat horizontal brain sections were stained using mouse monoclonal GFAP (HB8267) and rabbit monoclonal Neurofilament L (HB7266) antibodies from Hello Bio which were visualised using a Janelia Fluor® 549 SE conjugated goat anti-mouse antibody and a DyLight 488 conjugated goat anti-rabbit antibody. For more details see protocols #1 and #2 in the application notes below.
Fig 2. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody.
Figure 2. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody. Rat horizontal brain sections were stained using mouse monoclonal GFAP (HB8267) and rabbit monoclonal Neurofilament L (HB7266) antibodies from Hello Bio which were visualised using a Janelia Fluor® 549 SE conjugated goat anti-mouse antibody and a DyLight 488 conjugated goat anti-rabbit antibody. For more details see protocols #1 and #2 in the application notes below.
Fig 3. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody.
Figure 3. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody. Rat horizontal brain sections were stained using mouse monoclonal GFAP (HB8267) and rabbit monoclonal Neurofilament L (HB7266) antibodies from Hello Bio which were visualised using a Janelia Fluor® 549 SE conjugated goat anti-mouse antibody and a DyLight 488 conjugated goat anti-rabbit antibody. For more details see protocols #1 and #2 in the application notes below.
Fig4. Excitation and emission spectra of Janelia Fluor ® 525, SE.
Figure4. Excitation and emission spectra of Janelia Fluor ® 525, SE. Janelia Fluor® 525 is a yellow fluorescent dye suitable for confocal and super resolution microscopy. A 1 µM solution was prepared in PBS before excitation, emission and absorbance spectra were generated. For protocol see #Protocol 3 in application notes below.
Fig 1. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody.
Figure 1. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody. Rat horizontal brain sections were stained using mouse monoclonal GFAP (HB8267) and rabbit monoclonal Neurofilament L (HB7266) antibodies from Hello Bio which were visualised using a Janelia Fluor® 549 SE conjugated goat anti-mouse antibody and a DyLight 488 conjugated goat anti-rabbit antibody. For more details see protocols #1 and #2 in the application notes below.
Fig 2. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody.
Figure 2. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody. Rat horizontal brain sections were stained using mouse monoclonal GFAP (HB8267) and rabbit monoclonal Neurofilament L (HB7266) antibodies from Hello Bio which were visualised using a Janelia Fluor® 549 SE conjugated goat anti-mouse antibody and a DyLight 488 conjugated goat anti-rabbit antibody. For more details see protocols #1 and #2 in the application notes below.
Fig 3. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody.
Figure 3. GFAP expression in rat brain visualised using a Janelia Fluor® 525 conjugated secondary antibody. Rat horizontal brain sections were stained using mouse monoclonal GFAP (HB8267) and rabbit monoclonal Neurofilament L (HB7266) antibodies from Hello Bio which were visualised using a Janelia Fluor® 549 SE conjugated goat anti-mouse antibody and a DyLight 488 conjugated goat anti-rabbit antibody. For more details see protocols #1 and #2 in the application notes below.
Fig4. Excitation and emission spectra of Janelia Fluor ® 525, SE.
Figure4. Excitation and emission spectra of Janelia Fluor ® 525, SE. Janelia Fluor® 525 is a yellow fluorescent dye suitable for confocal and super resolution microscopy. A 1 µM solution was prepared in PBS before excitation, emission and absorbance spectra were generated. For protocol see #Protocol 3 in application notes below.
Add 10% by volume of 0.75M Tris-HCl pH7.4 (to a final concentration of 75mM) to stop the conjugation reaction. Incubate for 10-15 minutes at room temperature in the dark with gentle mixing.
Use a desalting column, perform buffer exchange (following manufacture instructions) of antibody into PBS 0.05% sodium azide. This step also removes any unbound dye
#Protocol 2: Immunohistochemistry
40μm horizontal sections were cut from a 4% PFA fixed rat brain.
