Microscopy Reagents for Immunocytochemistry and Immunohistochemistry

BioLegend provides a variety of reagents supporting microscopy-based imaging of cells and tissue samples. Our reagents include antibody conjugates to bright, photostable fluorophores like Brilliant Violet 421™, Brilliant Violet 510™ and the Alexa Fluor® dyes. We also provide cell tracking dyes, probes for cell health, subcellular localization and nuclear counterstains. BioLegend will continue to provide quality microscopy products as we discover innovative technologies.


Watch our full step-by-step protocol videos on FFPE and Frozen Tissue Processing, and staining with Immunofluorescence and Chromongenic DAB Detection Systems.

Purified and Biotin-labeled Primary Antibodies

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.

Spark YG™ 570

Ideal for building multicolor microscopy panels, Spark YG™ 570 is a small, synthetic fluorophore that provides a bright and photostable signal. With an excitation max of 555 nm and an emission max of 570 nm, it can be imaged using the same filter sets commonly used for Alexa Fluor® 555, Cy3, or TRITC. Spark YG™ 570 can be used in either widefield or confocal microscopy. Depending on the filter sets and light source of your microscope, Spark YG™ 570 can be used in combination with a variety of other fluorophores including DAPI, Alexa Fluor® 488, Alexa Fluor® 647, and Alexa Fluor® 750.


C57BL/6 mouse frozen spleen section was fixed with 4% paraformaldehyde for 10 minutes at room temperature and blocked with 5% FBS. Then the section was stained with 5 µg/mL of CD45 (clone 30-F11) Spark YG™ 570 (red), CD3 (clone 17A2) Alexa Fluor® 647 (green), and B220 (clone RA3-6B2) Alexa Fluor® 488 (blue) overnight at 4°C. The image was captured with a 10X objective.

Alexa Fluor® 488, Alexa Fluor® 594 and Alexa Fluor® 647

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™, Brilliant Violet 510™

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. 

Secondary Reagents

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.

In addition to primary antibodies, a number of dyes or probes can be used to specifically stain subcellular structures. These can be used across a wide variety of microscopy applications, including counterstaining the nucleus or identifying general cytoplasmic structure. Explore our available cell stains below.

Please select a stain.

Nucleic Acid Stains

Permeant and impermeant nucleic acid stains are an excellent tool for visualizing the location and number of cells in a sample and providing spatial context for the antigens of interest. In a live-cell imaging application, an impermeant nucleic acid stain like DRAQ7™Helix NP™ BlueGreen or NIR can be paired with a permeant nucleic acid stain like DAPICytoPhase™ Violet, or DRAQ5™ to assess the live to dead cell ratio.

Learn more about Non-Antibody Chemical Probes.

Actin Stains

Phalloidin is a bicyclic peptide found in death cap mushrooms that binds very tightly to F-actin, preventing its depolymerization in living cells. In cellular imaging, fluorescently conjugated phalloidins are useful for imaging the fine filaments of actin, providing structural and volumetric context to the cell. BioLegend offers our proprietary probes Flash Phalloidin™ Red 594, and Green 488 for identifying F-actin structures.

Learn more about Non-Antibody Chemical Probes.

Mitochondrial Stains

Fluorogenic chemical reagents that are attracted to the polarization of the mitochondrial membrane make ideal probes for imaging mitochondrial localization in microscopy. Initially, the cells must be live while labeling, getting excellent signal at low concentrations. However, MitoSpy™ probes, like MitoSpy™ Orange CMTMRos and MitoSpy™ Red CMXRos, can be retained with an aldehyde-based fixative when incubated at higher concentrations of the probe in order to be combined with intracellular antibody detection. MitoSpy™ Green FM and MitoSpy NIR™ DiIC1(5) are only useful for useful for mitochondrial localization in live cells. 

Learn more about MitoSpy™ Probes.

Long Term Protein Stains for Cell Tracking and Proliferation

CFDA-SE (CFSE), and Tag-it Violet™ are cell permeant protein stains that can be used for cell proliferation and tracking in microscopy applications. Zombie Dyes are reagents that label all cells through a covalent attachment to cell surface amine-containing proteins, offering a long-term cell tracking alternative without intracellular delivery or cytotoxicity. Cells can proliferate uninhibited until the signal intensity becomes too diluted. 

Learn more about Non-Antibody Chemical Probes.

Reagents for Chromogenic IHC

Chromogenic detection methods have a long history in histology and pathology applications. Commonly used in chromogenic IHC are antibodies or streptavidin covalently attached with HRP or AlkPhos, that convert a substrate like DABAEC, or BCIP/NBT. These enzymes catalyze their substrates, leaving a deposit of color where the antibody has attached to the cell or tissue. BioLegend provides a variety of accessory reagents for performing IHC including the Retrieve-All Antigen Unmasking SystemUltra-Streptavidin (USA) HRP Detection Kits, and ACUITYAdvanced Biotin Free Polymer Detection Kits

View all buffers and detection kits.

Biological tissues are generally composed of proteins, lipids, and water, each of which has a different refractive index (RI). Differences in refractive indices, or RI mismatch, cause the scattering of light in the tissue and result in tissue opacity. Our Ce3D™ Tissue Clearing Solution reduces light scattering by normalizing the RI throughout the tissue, thus making the tissue transparent. This reagent allows for rapid clearing of tissues and the generation of mesmerizing 3D images in multicolor fluorescence microscopy applications.


To learn more, visit our Ce3D™ Tissue Clearing Webpage.


3D IHC and Tissue Clearing

Generating the best image involves many factors, all dependent on what is ideal for imaging the marker of interest in its biological context. Here are some questions to ask yourself when choosing the right reagents and instruments.

Do I want to image tissue thicker than 10 µm? Confocal or Multiphoton Microscopy
Do I want to image more than 4 colors on a cell sample? Spectral Unmixing
Do I want to reconstruct the sample in 3D? Confocal or ApoTome
What level of resolution is desired/required? Deconvolution, Structured Illumination, STED or PALM
Do I want to demonstrate colocalization/binding or bioactivity in live cells? FRET or FLIM

1. Number of Targets

Using the spectral data of each fluorophore, you can make choices about optimal filter selection to minimize spectral spillover in experiments up to 4 colors for both confocal and widefield microscopy. Above 5 colors, a microscope employing spectral detection becomes useful to unmix the spectral spillover. If using antibodies for detection, problems can arise with the species-dependence of the primary and secondary antibody combination. Ideally, the use of directly labeled antibodies or haptens like biotin/streptavidin can help.


2. Fluorophore Combinations with Overlapping Spectra

One tip when using fluorophores where one spills into the other is to make sure the two antibodies are not imaged on markers that co-localize. For example, image one on a marker in the nucleus and the other at the cell surface, when possible. Also, make sure the fluorophore that is spilling over into the neighbor filter is on the less abundant antigen.

3. GFP or a fluorescent protein variant

Fluorescent proteins do not survive exposure to methanol or acetone. If the GFP signal was present prior to fixation but signal is lost upon fixation, check to see if the paraformaldehyde was reconstituted with the help of methanol. If the fixative can’t be changed to be organic solvent-free, anti-GFP antibodies can be employed to recover the GFP signal.

4. Instrument Choice

The instrument is made to be an ideal tool for the biological question, not the reverse. The better you understand the goal of the image, the better you can match the application to the instrument.

5. Sensitivity

Sensitivity is a balance between the signal strength and non-specific staining/autofluorescence/background. Biological autofluorescence will be endogenous in certain tissues, like brain, liver, lung, etc. The following can be applied to minimize background and improve signal strength:

  • An appropriately complex blocking step, for example serum instead of BSA or milk, prior to adding antibodies.
  • If streptavidin is used and the tissue will be fixed and permeabilized, an endogenous biotin-blocking kit can prevent the biotin found naturally in mitochondria from binding the streptavidin.
  • Use directly conjugated primary antibodies for sufficiently abundant antigens.
  • Use secondary antibodies or other amplification methods for lowly expressed antigens. Another option is to use biotin and streptavidin or other hapten-based amplification methods.
  • Use enzymatic amplification kits like tyramide signal amplification (TSA) kits for extremely lowly expressed antigens. As a cautionary note, amplification and secondary techniques will also likely increase your background.

6. Antifade

Finally, mounting media containing antifade is required for the maintenance of signal strength. All organic fluorophores photobleach, a process where reactive oxygen species created in the process of imaging attack the structure of the fluorophores, irreversibly neutralizing their ability to fluoresce. Using antifade is more difficult when the cells are imaged live, since any antifade scavenges oxygen from the media, thus suffocating the cells. This is why regenerating signal, like proteins expressing GFP, are desirable for long-term, live-cell imaging.

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