Collagen Hybridizing Peptide Staining

Collagen Hybridizing Peptide (CHP) staining is a method used in histology and tissue engineering to visualize and quantify collagen fibers in biological samples. CHP staining works by using a peptide probe that specifically binds to the type I collagen fibers, which are the most abundant type of collagen in many tissues. The bound probe is then visualized using a secondary antibody conjugated to a fluorescent label, allowing for the detection and quantification of collagen fibers in tissues and cell cultures. This staining method is useful for a variety of applications, including the analysis of tissue morphology, the assessment of tissue health and viability, and the study of collagen-based materials in tissue engineering.

Collagen Hybridizing Peptide

In mammals, Collagen is the most abundant protein and the main structural component of all organs and tissues. It is the main building block of  tendon, ligament, cornea, cartilage, and bone.


The triple helical structure of Collagen can undergoes unfolding in tissue during diseases, development, mechanical injury, and decellularization. The collagen hybridizing peptide (CHP) binds with these unfolded collagen chains, both in vitro and in vivo, and help us in the detection of inflammation and tissue damage which are caused by different types of disease, as well as tissue remodeling.

Traditional techniques are tough to identify for these varieties of collagen degradations and therefore a planned peptide (Collagen Hybridizing Peptide, CHP), that hybridizes the degraded collagen was created.


It is a synthetic peptide and contain a 6 to 10 repeating units of Gyl-Xaa-Yaa amino acid triplet, which mimicking the natural collagen sequence. The CHP has a high tendency to form the triple helix conformation, because of the presence of proline and Hydroxyproline in the Xaa-Yaa position.

The monomeric peptide chain forms a hybridized triple helix with the collagen strand which help  to recognize denatured collagen strands in body tissues. The triple-helical chain ( Gly-Xaa-Yaa) and hydrogen bond interchains help to enable this mechanism.


Due to a lack of binding sites, the Collagen Hybridizing Peptide is very precise with a negligible affinity to bind collagen molecules.

Applications of Collagen Hybridizing Peptide

  • Used for Immunofluorescence.
  • Used for Immunohistochemistry
  • Used for cell imaging (2D or 3D)
  • Used for SDS-PAGE (in-gel Western blot).

Features of CHP reagents

  • More informative, reliable and convenient than zymography, DQ collagen, SHG, and TEM
  • High affinity and unparalleled specificity to collagen with essentially no nonspecific binding
  • Applicable to denatured collagens of all subtypes and from all species; binding relying on collagen’s secondary structure instead of specific epitopes
  • Suitable for both frozen and paraffin-embedded sections with no need for antigen retrieval
  • A non-antibody approach with no species restrictions, compatible for co-staining with any antibody
  • Small size (2% of IgG by MW) enabling facile tissue penetration during whole tissue staining (with no need for sectioning)
  • Stable in solution under 4 °C, eliminating the need to aliquot for storage

Collagen Hybridizing Peptide Staining


Collagen Hybridizing Peptide Staining is used to detect the Denatured collagen ligaments.


Collagen Hybridizing Peptide Staining Principle

Collagen Hybridizing Peptide stain is a vital stain used for the detection of collagen degraded tissues, mainly used in  Developmental Biology, Histology, and Histopathology.

The cellular matrix degradation by proteolytic migration of inflamed cells within a collagen culture is visualized by CHP stain. It also can quantify the local mechanical injuries at a molecular level including assessing the denaturation of the collagen tissues in the extracellular matrix level. 


In SDS-PAGE it helps us to visualize the collagen bands without the application of the western blotting method. When CHP solution remains in stored condition it slowly reassembles into triple helices, therefore unfolded collagen strands can not hybridize it.

Before use, heat must be applied to separated into monomers. The formation of trimers  (trimerization) by CHP needs some time even hours when in low concentrations because the heat-dissociated CHP can stay as an active strand for hybridizing the denatured collagen.


Heating the CHP at 80 °C in a water bath after dilution is ideal common protocol is heating the CHP solution (after diluting to the desired concentration) to and quickly quenching it to room temperature followed by immediate application to target collagen substrates, as described below in detail. A heating block and an ice-water bath may be needed in most applications (not provided).

Practically, CHP can be labeled with biotin for avidin/streptavidin-mediated detection

Reagents Required

  • Peptide powder
  • Phosphate buffered water
  • Biotin
  • Basal Salt

Collagen Hybridizing Peptide Staining Procedure

Biotin CHP (Chemically PEGylated Horseradish Peroxidase) is a popular reagent used for staining tissues for research purposes. This article provides a comprehensive guide to preparing and using Biotin CHP for staining tissue samples.

1. Preparing the Stock Solution

  • Dissolve 0.3 mg of Biotin CHP powder in 1 mL of pure water or PBS (phosphate-buffered saline).
  • Mix well and centrifuge to create a stock solution with a minimum concentration of 100 μM.
  • Store the stock solution at 4°C.

2. Diluting the Stock Solution

  1. Dilute the stock solution to the desired concentration depending on the tissue being tested. For example, dissolve 60 μg of powder in 400 μL of water or PBS to get a 50 μM stock solution.

3. Blocking the Tissue Sample

  1. Block the tissue sample with 10% serum or 5% BSA, particularly when co-staining with antibodies.
  2. In some tissue types, such as the kidney, it may be necessary to block endogenous biotin using a standard kit for B-CHP staining.

4. Heating and Cooling the CHP Solution

  • Dilute the CHP stock solution in PBS buffer with an average dilution of the sample.
  • Heat the diluted CHP in a water bath at 80°C for 5 minutes.
  • Immediately place the heated CHP in a microtube in an ice water bath for 15-90 seconds to avoid damaging the tissue sample with heat.
  • Centrifuge the cooled microtube to collect the condensation.
  • Pipet the solution to each tissue sample.

5. Incubating the Tissue Samples

  • Incubate the tissue samples with the staining solution at 4°C for 2 hours or overnight for better results.

6. Washing the Tissue Slides

  1. Wash the tissue slides in PBS for 5 minutes in three changes at room temperature.

7. Analyzing the Stained Samples

  • CHP and R-CHP can be analyzed with a fluorescence microscope.
  • B-CHP can be detected by an avidin/streptavidin-mediated method.

Conclusion Staining tissue samples with Biotin CHP is a crucial step in many research studies. By following this step-by-step guide, researchers can prepare and use Biotin CHP effectively to obtain optimal results.

Result and interpretation of Collagen Hybridizing Peptide Staining

Result and interpretation of Collagen Hybridizing Peptide Staining
Sagittal segment of E18 mouse embryo showing localization of CHP binding using B-CHP (orange, detected by AlexaFluor647-streptavidin) and an anti-collagen I antibody (detected by AlexaFluor555-labeled donkey anti-rabbit IgG H&L, cyan). The maxilla, mandible, sphenoid, occipital, spinal column, rib cage, hip bone, and fingers are all represented here.
  • For F-CHP: observe green stained separated porcine ligament cryosections.
  • For R-CHP: the heated ligaments will appear red, while the intact ligaments appeared blue, and the unheated ligaments dark blue (Black).
  • For B-CHP: the ligaments appear light brown.

Advantages of Collagen Hybridizing Peptide Staining

  • Has a specificity for denatured monomeric collagen strands.
  • It has a high sensitivity for detection of denatured collagen ligaments.

Disadvantages of Collagen Hybridizing Peptide Staining

Importance of Collagen Hybridizing Peptide Staining

  • It can be used for other species of organisms.
  • Used to differentiate collagen types.
  • Used to detect the inflammation and tissue damage.
  • Used to detect embryonic bone deformation.
  • Used to detect skin aging.
  • Used to detect osteoarthritis, pulmonary fibrosis, myocardial infarction, glomerulonephritis.



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