Cell-permeable, yellow fluorescent dye with an NHS ester (succinimidyl ester (SE)) reactive group. NHS esters react with primary amines on proteins and are commonly used for conjugating dyes to proteins, antibodies amine-modified oligonucleotides etc.
Suitable for super resolution microscopy (SRM) including techniques such as dSTORM (both live and fixed cells), cellular imaging when combined with the HaloTag or SNAP-tag self labelling systems and confocal microscopy. 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.
