IHC/ICC Applications

MicroscopyIHC/ICC Applications

IHC/ICC Applications

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Immunocytochemistry (ICC) is characterized by imaging primary cells or cell lines in culture. Immunohistochemistry (IHC) refers to the detection of antibodies in tissue sections. Both ICC and IHC applications can utilize chromogenic or fluorescent detection methods. On our product technical data sheets, we use IHC-P to indicate that the antibody is useful in formalin-fixed paraffin-embedded (FFPE) sections and IHC-F to indicate the antibody is useful in tissue that has been fixed and frozen prior to sectioning. If there is only an IHC designation, check the literature citations to determine the method of tissue preparation compatible with each reagent.

View our IHC and ICC protocol videos for step-by-step guides to different microscopy workflows.

Newly Validated IHC Antibodies

Our scientists are continuously working to update application validation to our extensive clone library. Explore our newly validated IHC antibodies for cell and tissue imaging to drive discovery in various research fields, including oncology, immunology, and neurobiology.

View our recent antibody clones validated for IHC.

Fluorophores for Microscopy

Fluorescent detection allows visualization of multiple markers at a time, most commonly through the use of discrete excitation sources optimal for each fluorophore. Fluorescent detection introduces the opportunity for advanced imaging applications as well, like live-cell imaging, multiphoton imaging, super-resolution microscopy, FLIM, and FRET.

Although sensitivity can be a limitation of fluorescence microscopy at certain wavelengths, especially reagents that emit in the range of 350-450 nm due to increased autofluorescence of the sample, improved signal-to-noise can be obtained through varying enzymatic and immunologic amplification techniques, the use of higher sensitivity instrumentation, and near-infrared emitting fluorophores.

Right: Paraffin-embedded human prostate tissue was stained with anti-CD44 Alexa Fluor® 594 (red), purified anti-human CD107b (H4B4) antibody followed by anti-mouse IgG Alexa Fluor® 488 secondary antibody (green) and DAPI (blue).

We provide a vast selection of fluorophore antibody conjugates for your microscopy research. The table below highlights some of these conjugates and their emission profiles.

Direct Conjugate	Excitation max (nm)	Emission max (nm)	Emission color
DyLight™ 405	400	420	Blue
Brilliant Violet 421™	405	421	Blue
Brilliant Violet 510™	405	510	Green
DyLight™ 488	493	518	Green
Alexa Fluor® 488	495	519	Green
Alexa Fluor® 555	555	565	Orange
Spark YG™ 570	555	570	Orange
Alexa Fluor® 594	590	617	Red
DyLight™ 594	592	617	Red
Alexa Fluor® 647	650	665	Near-IR*
Alexa Fluor® 700	702	723	Near-IR*

* Human vision is not sensitive to light beyond ~650 nm; it is not possible to view near-IR fluorescent dyes.

Alexa Fluor® Dyes

Alexa Fluor® dyes are well-described probes with consistent output in the field of microscopy. While Alexa Fluor® 488, Alexa Fluor® 594, and Alexa Fluor® 647 are the most commonly used fluorophores in microscopy, additional family members like Alexa Fluor® 555 and Alexa Fluor® 700 can also provide strong and photostable staining for imaging.

View all Alexa Fluor® products >

Brilliant Violet™ Fluorophores

Brilliant Violet 421™ (BV421™) is used in the “blue” channel which is typically occupied by DAPI or Alexa Fluor® 405. Brilliant Violet 510™ (BV510™) is also excited at 405 nm, but emits at 510 nm. When your filter set-up is optimized, these organic polymers, BV421™ and BV510 ™, can be used simultaneously as bright, photostable options for multicolor microscopy.

Learn more about Brilliant Violet™ >

Spark YG™ 570

Ideal for building multicolor microscopy panels, Spark YG™ 570 stands out as a reliable addition to any multicolor microscopy panel. With an excitation max of 555 nm and an emission max of 570 nm, it can be imaged using the filter sets commonly used for Alexa Fluor® 555, Cy3, or TRITC in either widefield or confocal microscopy.

View our Spark YG™ 570 Products >

Five-Color Fluorescence Microscopy

With increased fluorophore options and the abundance of directly conjugated antibodies, it is possible to use fluorescence microscopy to look at 5 different markers on a single sample. For larger microscopy panels, it is especially critical to know your microscope and be sure it has the appropriate lasers and filters to capture the emission and excitation spectra of each of these distinct fluorophores.

We stained frozen C57BL/6 mouse spleen tissue using antibodies against CD4 and CD8a to detect T cells, B220 to stain B cells, and CD169 and F4/80 to detect tissue-resident macrophages. For this staining, we used antibodies directly-conjugated to bright photostable fluorophores including the Brilliant Violet™ and Alexa Fluor® dyes.

fullstaining

Microscope Excitation and Emission Filters

By optimizing your filter set-up to allow for the detection of Brilliant Violet 421™ and Brilliant Violet 510™, you can expand your microscopy panels. See how we optimized our set-up below.

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. We provide hundreds of purified antibody products and dozens of biotinylated antibodies, quality-tested for ICC or IHC.

View our Purified Antibodies and Biotinylated Antibodies.

Secondary Reagents

Signal amplification is often required in imaging applications for lowly expressed antigens. One way to increase the likelihood of success when imaging a target is to amplify the primary 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 a selection of dye-conjugated secondary reagents.

Explore our Secondary Reagents and Streptavidin Conjugates.

Chemical Probes

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 staining of the nucleus, organelles, or cytoplasmic structures, such as actin and microtubules.

Our table highlights a number of our non-antibody chemical probes, their subcellular localization, and emission profiles.

Chemical Probe	Subcellular Localization	Excitation Max (nm)	Emission Max (nm)	Emission Color
Tag-it Violet™	Cytoplasm	405	450	Blue
DAPI	Nucleus	360	460	Blue
Helix NP™ Blue	Nucleus	430	470	Blue
MitoSpy™ Green FM	Mitochondria	490	516	Green
Helix NP™ Green	Nucleus	495	519	Green
Flash Phalloidin™ Green 488	Actin	488	520	Green
Apotracker™ Green	Phosphatidylserine	500	520	Green
Calcein Red-AM	Cytoplasm	560	574	Orange
MitoSpy™ Orange CMTMRos	Mitochondria	551	576	Orange
MitoSpy™ Red CMXRos	Mitochondria	577	598	Red
Flash Phalloidin™ Red 594	Actin	590	611	Red
MitoSpy™ NIR DilC1(5)	Mitochondria	638	658	Near-IR
Helix NP™ NIR	Nucleus	640	660	Near-IR
DRAQ5™	Nucleus	633	695	Near-IR

Reagents for Chromogenic Detection

Chromogenic detection methods are advantageous because a signal can be amplified simply by extending the amount of time and substrate in the reaction. Also, it does not require sophisticated instruments for detection, only a microscope with phase contrast. HRP detection can, however, be accompanied by endogenous background associated with cellular peroxidase activity, non-specific signal, and is only typically used to image a single marker at a time.

These 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 DAB, AEC, or BCIP/NBT. These enzymes catalyze their substrates, leaving a deposit of color where the antibody has attached to the cell or tissue. We provide a variety of accessory reagents for performing IHC including, Ultra-Streptavidin (USA) HRP Detection Kits, and ACUITYAdvanced Biotin Free Polymer Detection Kits.

IHC staining of anti-DJ-1 antibody (PARK7) on FFPE normal (left) and Parkinson’s disease (right) brain tissue. Ultra Streptavidin (USA) HRP Detection Kit was used for detection followed by hematoxylin and bluing solution counterstaining. Scale bar: 50 µm.

IHC/ICC Applications

Fluorophores for Microscopy

Alexa Fluor® Dyes

Brilliant Violet™ Fluorophores

Spark YG™ 570

Five-Color Fluorescence Microscopy

Microscope Excitation and Emission Filters

Purified and Biotin-Labeled Primary Antibodies

Secondary Reagents

Chemical Probes

Reagents for Chromogenic Detection

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