Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is a technique commonly used for morphological characterization of tumors or other tissue malignancies. IHC uses antibodies to detect and analyze protein expression while maintaining the composition, cellular characteristics, and structure of native tissue. We offer IHC validated antibodies and detection systems. 

Immunohistochemistry

Antigen retrieval

Crosslinks created during the fixation step can prevent antibody binding by inhibiting access to the antigen; therefore, it is important to reverse crosslinks using a procedure called antigen retrieval (also known as antigen unmasking or epitope retrieval). Antigen retrival can be achieved through either a heat-induced method (heat-induced epitope retrieval; HIER) or through enzymatic digestion. 

Heat-induced epitope retrieval (HIER)

There are multiple buffers available for HIER. The two commonly recommended are citrate buffer and EDTA buffer. The appropriate buffer for your experiment will depend on the primary antibody you are using. Thus, always refer to your product datasheet for the recommended retrival buffer for your specific antibody. 

We offer these buffer alternatives:

H-3300-250 Antigen Unmasking Solution, Citrate-Based Datasheet
H-3301-250 Antigen Unmasking Solution, Tris-Based Datasheet
14746 SignalStain Citrat unmasking løsning Datasheet
14747 SignalStain EDTA unmasking løsning Datasheet
Enzymatisk antigen retrieval

Antigen retrieval can also be achieved through enzymatic digestion with pepsin, trypsin, or proteinase K. For those antibodies that require enzymatic retrieval rather than HIER, the recommended enzyme and digesting conditions will be clearly indicated on the product datasheet. 

 

It is important to note that antigen retrieval requirements are specific for the unique antibody/antigen and not for the protein itself. Therefore, if you use more than one antibody against a particular protein target, the optimal retrieval conditions for each antibody must be determined individually. 

Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Antibodies for IHC

IHC identifies the presence and pattern of expression of proteins in a biological sample through specific antibody binding. The quality of the primary antibody is very important in an IHC set up. Antibodies with low specificity and poor validation may result in weak signal and/or high background. We offer a wide range of IHC-validated antibodies which will give you clear good results. 

Popular antibodies:
• ROS1 (D4D6®) Rabbit mAb (#3287)
• ALK (D5F3®) XP® Rabbit mAb (#3633)
PD-L1 (E1L3N®) XP® Rabbit mAb (#13684)
• TIM-3 (D5D5R™) XP® Rabbit mAb (#45208)
• PD-L2 (D7U8C™) XP® Rabbit mAb (#82723)
• PRAME (E7I1B) Rabbit mAb (#56426)
• F4/80 (D2S9R) XP® Rabbit mAb (#70076)
• PD-1 (D4W2J) XP® Rabbit mAb (#86163)
• CD8α (D4W2Z) XP® Rabbit mAb (Mouse Specific) (#98941)
• CD31 (PECAM-1) (D8V9E) XP® Rabbit mAb (#77699)
• FoxP3 (D6O8R) Rabbit mAb (#12653)
• CD4 (D7D2Z) Rabbit mAb (#25229)

Antibodies for IHC

Our supplier Cell Signaling Technology (CST) is known for their extensive validation of antibodies for different applications. The determination of target specificity in IHC analysis requires multiple validation steps. CST scientists use
a variety of methods, as appropriate, to validate each IHC recommended antibody. 
Read more about how they do the validation here: IHC Validation at CST  

Check out this list of IHC-validated antibodies for cancer immunology research:
IHC-Validated Products for Cancer Immunology Research
 

IHC detection systems

Detection systems for immunohistochemistry (IHC) - both ABC (avidin-biotin complex) and polymer-based detection systems are available. The ABC kits are available as either peroxidase- or alkaline phosphatase-based.  Find the systems best suited for your samples and get the strongest signals with the lowest background.  

...  Learn more   
IHC detection systems

Counterstaining

Counterstaining aids in visualization and target localization, facilitating interpretation of morphology and cell structure within the tissue section. 

Counterstains

Our nuclear counterstains from Vector laboratories are packaged as convenient, ready-to-use solutions for use on individual slides or in staining dishes.
 

Counterstaining

Vector® Hematoxylin (blue)
Based on Gill's III fomulation. Progressive stain formula, thus the intensity can be adjusted to optimize results for either manual or automated systems. Gives excellent color contrast with most commonly used peroxidase and alkaline phosphatase substrates. It is suitable for use with non-aqueous and aqueous mounting media. Alcohol- and mercury-free. 

Vector® Hematoxylin QS (blue)
A modification of Mayer's hematoxylin developed especially for immunocytochemistry (ICC). Stains in less than 45 seconds, with excellent color contrast with most commonly used peroxidase and alkaline phosphatase substrates. Suitable for use with non-aqueous and aqueous mounting media. Mercury-free. 

Vector® Methyl Green (light green)
A superior formulation of methyl green suitable for use with a wide range of enzyme substrates. A simple, two-step procedure, and it is an excellent alternative in multiple antigen labeling when hematoxylin obscures the substrates colors. Suitable for use with non-aqueous mounting media. 

Vector® Nuclear Fast Red (pink)
A fast one-step protocol. This is also an excellent alternative in multiple antigen labeling when hematoxylin obscures the substrates colors. Gives good contrast with a variety of substrates. 

Hematoxylin and Eosin Stain Kit
This kit includes a modified eosin for staining of cytoplasm and hematoxylin for sharp, intens staining of the nucleus.

Special stains

Use Alcian Blue (pH 2,5) Stain Kit from Vector laboratories for histological visualization of sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins (glycoproteins).

• Acidic Sulfated Mucosubstances: Blue
• Hyaluronic Acid: Blue
• Sialomucins: Blue
• Nuclei: Red

Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Mounting

Slides should be mounted with coverslips for specimen preservation and optimal viewing. Aqueous and nonaqueous (permanent) mounting media are available. the mounting media you choose depends on the chromogen used during the detection step and its solubility in organic solvents or water; alcohol- or xylene-soluble chromogens should be used with nonaqueous mounting media and water-soluble chromogens should not be used with aqueous media. 

Here are some examples of commonly used peroxidase-based chromogens and corresponding mounting media: 

Chromogen Color Mounting media
DAB Brown Nonaqueous or aqueous
AEC Red  Aqueous
NovaRedTM Red Nonaqueous
There are many other commercially available peroxidase-based chromogens. Always consult the manufacturer for the recommended mounting media. 
 
Aqueous mounting media

SignalStain® Mounting Medium from Cell Signaling Technology (CST), permanently preserves tissue or cell preparations, and produces clear mounted sections, free from crystallization, with the ideal refractive index for high-resolution oil immersion microscopy. SignalStain® Mounting Medium retains the staining intensity of slides prepared with f.ex. SignalStain® DAB Substrate Kit #8059.

14177 SignalStain® Mounting Medium Datasheet

VectaMount AQ Mounting Medium from Vector Laboratories, is a hardsetting mounting medium developed for use with enzymatic substrates, such as AEC, ImmPACT® AEC, and ImmPACT AMEC Red, whose reaction products are soluble in alcohol or other organic solvents. In applications where aqueous mounting is preferred, VectaMount AQ is suitable for use with other substrates such as DAB, Vector® SG, BCIP/NBT, Vector Red, ImmPACT Vector Red, Vector Blue, ImmPACT DAB, ImmPACT DAB EqV, and ImmPACT SG.

H-5501-60 VectaMount® AQ Aqueous Mounting Medium Datasheet

 

Non-aqueous mounting media

VectaMount Mounting Medium from Vector Laboratories, is an optically clear and odorless formula for permanently preserving histochemical stains or precipitable enzyme substrates in tissue sections or cell preparations. It is compatible with horseradish peroxidase (HRP), alkaline phosphatase (AP), and glucose oxidase substrates. VectaMount Mounting Medium contains no toluene or xylene. 

H-5000-60 VectaMount® Permanent Mounting Media  Datasheet
Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

IHC control slides

We offer a wide range of control slides from Cell Signaling Technology (CST).

Example: Control slide for PD-L1 staining with formalin fixed, paraffin-embedded cell pellets, HDLM-2 (PD-L1 positive) and PC-3 (PD-L1 negative). 

Picture: Immunohistochemical analysis of paraffin-embedded HDLM-2 (left) and PC-3 (right) cell pellets using PD-L1 (E1L3N™) XP® Rabbit mAb #13684
IHC control slides
8117 SignalSlide® Phospho-ErbB Family IHC Controls Datasheet
8118 SignalSlide® Phospho-Met (Tyr1234/1235) IHC Controls Datasheet
55861 SignalSlide® HeLa -/+ IFNa IHC Controls Datasheet
13747 SignalSlide® PD-L1 IHC Controls Datasheet
41965 SignalSlide® Mouse PD-L1 IHC Controls Datasheet
13752 SignalSlide® LKB1 IHC Controls Datasheet
12873 SignalSlide® NF-κB p65 IHC Controls Datasheet
8104 SignalSlide® Cleaved Caspase-3 (Asp175) IHC Controls Datasheet
8101 SignalSlide® Phospho-Akt (Ser473) IHC Controls Datasheet
8103 SignalSlide® Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Controls Datasheet
8102 SignalSlide® Phospho-EGF Receptor IHC Controls Datasheet
8106 SignalSlide® PTEN IHC Controls Datasheet

Control slides may also be made on request. We deliver f.ex. control slides for ROS-1 to several laboratories. Contact us if you have needs that are not covered by the products in the list.

Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Multiplex IHC

Multiplex IHC enables a deeper understanding of co-expression and spatial organization of multiple targets within preserved tissue architecture, especially important when tissue samples are limited.

Ability to visualize multiple targets simultaneously is important in cancer immunology research, where it is necessary to catalog subsets of immune and cancer cells within the tumor microenvironment.

Multiplex IHC

In multiplex IHC, as well as in single or dual chromogenic IHC, using application-validated antibodies against relevant targets is crucial to obtaining reliable results. In addition, there are important protocol and technical considerations for selecting and using antibodies in multiplex assays.

Learn how to design a multiplex IHC assay in this application note:
Fluorescent Multiplex Immunohistochemistry with Tyramide Signal Amplification 

Examples of multiplex IHC assays are Fluorescent Multiplex Immunohistochemistry with tyramide or Imaging Mass Cytometry.

For Fluorescent Multiplex IHC with tyramide we recommend validated and specific antibodies for IHC-P. When it comes to Imaging Mass Cytometry we offer carrier-free antibodies to be used in higly multiplexed single-cell analysis and imaging.