Purified antibodies are the common go-to reagents for single color staining or in cases where secondary antibodies are desired for amplification. For amplification or multicolor applications, biotinylated antibodies are also effective tools. BioLegend provides over 900 purified antibody products and over 70 biotinylated antibodies, either quality-tested, validated, or reported in the literature for ICC or IHC.
Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647 are popular fluorophores that provide strong signal and are photostable for imaging. They can be used in confocal or widefield microscopy as long as the microscope has the required lasers and/or excitation and emission filter sets that suit the spectra of these fluorophores. BioLegend provides over 1600 antibodies conjugated to these Alexa Fluor® fluors.
C57BL/6 mouse frozen intestine section was fixed, blocked, and then stained with Alexa Fluor® 594 anti-mouse CD324 (clone DECMA-1) (red). Nuclei were counterstained with DAPI (blue).
Brilliant Violet 421™ (BV421™) and Brilliant Violet 510™ (BV510™) allow for expanded options in multicolor microscopy. BV421™ is used in the “blue” channel which is typically occupied by DAPI or Alexa Fluor® 405. BV510™ is also excited at 405 nm but emits at 520 nm. There are no other fluorophores that fit this particular spectral profile. Therefore, matching the excitation, emission and dichroic filters to this unique spectra is particularly important. However, when your filter set-up is optimized, BV421™ and BV510™ can be used simultaneously as bright, photostable options for multicolor microscopy.
C57BL/6 mouse frozen thymus section was stained with Brilliant Violet 421™ anti-CD80 (clone 16-10A1) (purple), Alexa Fluor® 647 anti-CD90.2 (clone 30-H12) (red) and Alexa Fluor® 594 anti-CD326 (clone G8.8) (green).
Vendors and Catalog numbers for BV421™ and BV510™ widefield filters:
| Chroma | Cat. No. 49027 | 49901 |
| Omega | Cat No. XF403 | XF438 |
| Semrock | Cat. No. BV421-3824A-000 |
Amplification is often required in imaging applications. One way to increase the likelihood of success when imaging a target is to amplify the primary intended signal without raising the background signal. In addition, amplifying a signal can also decrease the amount of exposure time, limiting the spillover of other fluors into your channel of interest. For these purposes, we provide many Alexa Fluor® and DyLight™ dye conjugated secondary reagents, including streptavidin and antibody products.
C57BL/6 mouse frozen spleen section was stained with Purified CD8b.2 (clone 53-5.8). The staining was followed by Alexa Fluor® 594 anti-Rat IgG1 (clone MRG1-58) (red) as a secondary, and Alexa Fluor® 488 anti-B220 (clone RA3-6B2 (green).