Collagen Hybridizing Peptides (CHPs) are short (30 amino acids in length), synthetic peptides specifically designed to hybridize to denatured or damaged collagen strands. They function through a structural mechanism: the CHP sequence recognizes and folds into a stable triple helix with unfolded individual collagen chains that have become exposed due to damage, degradation, or remodeling processes. CHPs are then conjugated to a fluorescence probe allowing for the visualization and assessment of collagen damage.

CHPs are engineered for high specificity towards damaged and denatured collagen molecules. They recognize the unfolded triple-helical collagen structure which occurs when collagen is damaged or undergoing remodeling. Under recommended experimental conditions, they exhibit minimal hybridization to intact, healthy collagen. This selectivity makes them ideal for detecting areas of collagen degradation or remodeling.

CHPs can bind to damaged collagen of all 28 subtypes within the collagen superfamily, regardless of species or tissue type. This broad reactivity is due to the highly conserved triple-helical structure of collagen that CHPs target once it becomes denatured.

• F-CHP: Labeled with fluorescein (5-FAM, green fluorescence, Ex/Em ~495 nm / ~520 nm). A popular choice for direct fluorescence detection in histology, immunofluorescence (IF), or in vitro assays.
• R-CHP: Labeled with sulfo-Cyanine3 (Cy3, red fluorescence, Ex/Em ~550 nm / ~570 nm). Functionally equivalent to F-CHP in binding effectiveness. Recommended for tissues with high autofluorescence in the green spectrum or when needing to avoid spectral overlap with other green markers.
• B-CHP: Labeled with biotin. Detected using avidin/streptavidin conjugated to a fluorophore or enzyme (e.g., HRP) of your choice. Offers flexible detection options, signal amplification, and can help avoid background noise.
• In vivo CHP: Labeled with sulfo-Cyanine7.5 (sCy7.5, near-infrared fluorescence). Incorporates our newest sequence, allowing direct injection into animals without a pre-activation heating step. Also usable for histology on tissue sections, though it is more expensive than CHPs optimized for histology.
• Auto-CHP: Specifically formulated for use in auto-staining platforms. Currently available for select partners. Please inquire if you are interested in this option.
• Scrambled Control Peptides (e.g., Scrambled R-CHP): These peptides have a randomized amino acid sequence and do not bind specifically to denatured collagen. They serve as a negative control to assess non-specific binding or background signal in your samples. Recommended for validating the specificity of CHP staining.

• R-CHP (Cy3): Typical excitation/emission maxima are ~550 nm / ~570 nm.
• F-CHP (5-FAM): Typical excitation/emission maxima are ~495 nm / ~520 nm.
• In vivo CHP (sCy7.5): This is a near-infrared dye. Please refer to the product datasheet for specific spectral characteristics.

No, we do not currently offer unconjugated CHPs as a standard product. Our Biotin-CHP (B-CHP) provides flexibility for users to employ their own streptavidin or neutravidin-conjugated labels (e.g., different fluorophores, enzymes) if our direct fluorescent labels are not suitable.

CHPs are chemically and physically very stable as a powder.

• Powder Form (Long-term): Store at -20°C.
• Reconstituted (In Solution): Store at 4°C.
• For F-CHP and B-CHP, there is no need to aliquot and freeze after reconstitution; they remain stable at 4°C through multiple heating/cooling cycles for preparing working solutions.
• For R-CHP and In vivo CHP, the attached dyes are less stable once reconstituted. These should be used immediately or aliquoted after reconstitution and stored at -20°C to reduce freeze-thaw cycles.
• Light Protection: R-CHP, F-CHP, and In vivo CHP solutions must be protected from light.

The 0.3 mg vial of CHP is typically reconstituted to create a 100 µM stock solution (e.g., by adding 1 mL of sterile water or PBS, but always verify based on the exact amount and MW on your vial).

• Precise Reconstitution: For precise applications, calculate the exact volume of buffer needed based on the specific molecular weight (MW) and the precise amount of CHP (e.g., in µg or nmol) provided on your vial label or product datasheet to achieve your desired stock concentration.
• Working Concentration: For most applications, a working concentration of 10 µM to 20 µM is a good starting point. Dilute the stock solution (e.g., 100 µM stock) 1:5 for 20 µM or 1:10 for 10 µM in PBS or an appropriate buffer. Some specific protocols or tissue types might benefit from optimization, potentially starting higher (e.g., 50-100 µM as mentioned in some user guide contexts). Always refer to the specific user guide for your CHP type and application.

Yes, for most CHPs (F-CHP, R-CHP, B-CHP), the peptide solution needs to be heated prior to each use each time you prepare the working dilution from the stock. This is to ensure the peptides are in their monomeric, single-stranded state, ready for hybridization. Once cooled and stored at 4°C in solution for a while, CHPs can gradually re-assemble into a trimeric form.

• Recommended Procedure: Aliquot the needed amount from the stock solution (e.g., 10 µL from a 50 µM or 100 µM stock), dilute it to the required working concentration, and then heat this diluted working solution (e.g., 80°C for 5-10 minutes or as per user guide) and cool to room temperature before applying to samples.
• Stability: After years of working with F-CHP and B-CHP, we found no limit to their heating/cooling cycles when this procedure is followed for the working solution. The peptide itself is very stable. We do NOT recommend repeatedly heating up the entire stock solution.
• Exception: In vivo CHP does NOT require this pre-activation heating step due to its modified sequence.

CHPs are versatile and suitable for various tissue processing methods, including:

• Frozen sections (e.g., OCT-embedded)
• Paraffin-embedded sections (FFPE)

Yes, heating during tissue processing (like paraffin embedding, typically 55-60°C) can cause some denaturation of collagen and may increase the baseline signal detected by CHPs. We recommend being as gentle as possible during processing. However, CHP will still effectively differentiate between regions of highly damaged collagen (e.g., due to disease or injury) and areas that may have been only mildly or uniformly affected by heat from processing. If you are concerned about heat-induced artifacts, consider using frozen/OCT embedded tissues as a comparison.

Fixation is not strictly necessary for CHP staining if the tissue architecture is otherwise preserved. However, if fixing is required for other experimental reasons (e.g., co-staining with antibodies, long-term preservation): * Chemical fixation can be performed beforehand. * For fluorescent imaging (e.g., when using F-CHP or R-CHP), we generally recommend using formaldehyde-based fixatives (e.g., 10% NBF, 4% PFA in PBS). Avoid paraformaldehyde or glutaraldehyde if you experience issues with autofluorescence, though many users successfully use PFA. Glutaraldehyde can significantly increase autofluorescence.

Temperature & Duration: Our standard protocols often suggest incubation at 4°C overnight for optimal binding. However, CHPs (such as F-CHP) also perform well at room temperature (e.g., 2 hours) or 37°C. If using a higher temperature like 37°C, the staining period will likely be shorter than an overnight incubation. If you have validated your protocol at 4°C, it should adapt well to 37°C with an appropriately adjusted (reduced) incubation time.

• Optimal Incubation: For optimal results, especially with FFPE sections or when maximum signal is desired, overnight incubation at 4°C is recommended. For In vivo CHP used on paraffin sections, overnight incubation is also recommended.

We typically recommend using about 100 µL of the diluted CHP working solution per standard tissue section, ensuring the section is completely covered to prevent drying out. With a 0.3 mg vial (yielding ~1 mL of 100 µM stock, assuming exact reconstitution):

• At a working concentration of 10 µM, you can prepare 10 mL of solution, sufficient for approximately 100 slides (at 100 µL/slide).
• At a working concentration of 20 µM, you can prepare 5 mL of solution, sufficient for approximately 50 slides (at 100 µL/slide).

If you observe very saturated pixels in your images, you can further dilute the CHP solution or reduce exposure time.

To create a positive control, you need to ensure there is abundant denatured collagen present. For tissue sections or collagen-containing hydrogels (e.g., rat tail collagen type I hydrogel):

• Heat the sample (e.g., slide with tissue section or hydrogel) at >80°C for 10-60 minutes (duration may vary depending on sample thickness and type).

Allow to cool, then proceed with the normal CHP staining protocol. This heat treatment will denature intact collagen, maximizing CHP binding and serving as a positive control for the CHP's ability to bind.

We typically recommend aqueous mounting media for fluorescently labeled CHPs to preserve fluorescence (e.g., VectaShield, ProLong Gold). We do not have direct extensive experience with Entellan or other non-aqueous/permanent mounting media. If you choose to use such a medium, ensure it does not have autofluorescent properties at the same wavelengths as your CHP and other fluorophores, and that it is compatible with the fluorescence of the dyes. We would appreciate hearing about your results if you try non-aqueous media, as it helps us advise other researchers.

Yes, a tissue slide can be readily co-stained with CHP and an antibody.

• Procedure: The primary antibody can often be directly diluted into the CHP working solution after the CHP solution has been heated and cooled down to room temperature. Then, co-stain the slides with the combined CHP and antibody solution, typically overnight at 4°C.
• Blocking: When co-staining, we recommend blocking the tissue slides with a general protein block (e.g., 10% normal serum of the secondary antibody host species, or 5% BSA) before applying CHP and antibodies.
• B-CHP Considerations: For certain tissue types (e.g., kidney, liver), it may be necessary to block endogenous biotin using a standard kit if using B-CHP and streptavidin-based detection.
• HIER: If Heat-Induced Epitope Retrieval (HIER) is required for your antibody, see Q5.2.

CHP staining should generally be performed after HIAR/HIER. The high temperatures used during antigen retrieval procedures will denature collagen and are likely to cause any pre-bound CHPs to dissociate.

• Option 1 (Recommended for detecting all exposed collagen after HIER): Perform HIAR/HIER first, then proceed with CHP staining. This approach will detect total collagen that has been denatured either by original tissue processing, disease state, or the HIER heat treatment itself.
• Option 2 (For detecting pre-existing denatured collagen without additional HIER-induced denaturation): If you want to assess only the collagen damage present before laboratory-induced denaturation by HIER, use a serial tissue section for CHP staining without performing HIAR/HIER on that specific section.
• Option 3 (Advanced Technique for co-staining): If using B-CHP, Tyramide Signal Amplification (TSA) might offer a way to detect signal even if CHP staining is performed before HIER, though this is an advanced technique. Some researchers have reported success using standard TSA kits for this purpose. For most antibodies requiring HIER, applying CHP after HIER and primary/secondary antibody incubation is a safer approach. If co-staining with CHP and an antibody that requires HIER, and you are detecting CHP directly (F-CHP, R-CHP), perform HIER, then antibody staining, then CHP staining.

CHPs can be used to detect total collagen by first intentionally denaturing all collagen in the sample. This is typically achieved by heating the tissue section on a slide (e.g., 80°C for 1 hour for paraffin sections, or as per specific total collagen protocols). This "unravels" intact collagen molecules, allowing CHPs to bind to all collagen present. Compared to traditional histological stains like PSR or Masson's Trichrome, CHPs offer several advantages for total collagen assessment:

• Specificity: CHPs bind specifically to collagen, unlike PSR or Trichrome which can sometimes bind non-specifically to other structures.
• Quantitative Analysis: CHP staining intensity (e.g., via immunofluorescence) can be more robustly quantified than the area fraction analysis often used with PSR/Trichrome, allowing for more nuanced measurements.
• Versatility: CHPs can be used alongside other IHC/IF markers for multiplex analysis.

For an example of CHPs used for total collagen (via heat denaturation) co-stained with a collagen I antibody, see our publication with Roche: Nature Communications Biology (2024).

While CHPs directly bind only to denatured collagen, you can use a sequential staining approach to infer regions of initially intact collagen:

1. (Optional) Induce specific damage if your model requires it (e.g., in cell culture).
2. Stain with CHPs (Color 1): This will visualize the initially denatured/damaged collagen. Acquire images.
3. Heat the sample: Denature all remaining intact collagen on the slide (e.g., 100°C for 20 min for paraffin sections, or as per total collagen protocol).
4. Re-stain with CHPs (Color 1 again, or Color 2 if using a different CHP type and imaging capabilities allow): This will visualize total collagen.

Analysis: The difference in signal intensity or area between step 4 and step 2 can then be attributed to the collagen that was initially intact but subsequently denatured by heating. Developing a robust simultaneous co-staining protocol for intact and denatured collagen (e.g., using CHPs with another probe specifically for intact collagen) is an area of interest but has been challenging to achieve with consistent, easily quantifiable success.

Yes, fluorescently labeled CHPs (e.g., F-CHP, R-CHP) can be utilized to stain collagen bands directly in SDS-PAGE gels after electrophoresis. This method can specifically identify collagen products within protein solutions or tissue lysates, offering an alternative to non-specific protein stains like Coomassie blue. The choice of lysis buffer would depend on your tissue and overall experimental goals, rather than specifically on the CHP staining step post-electrophoresis.

It is probable that CHPs (e.g., B-CHP) will bind to acid-hydrolyzed soluble Type I collagen, provided two conditions are met:

1. The pH is suitable for CHP binding (ideally pH 7-8).
2. The collagen fragments are sufficiently long (generally larger than ~2000 Da, meaning they can still form a partial triple helix with CHP).

Since acid-hydrolyzed collagen typically falls within a size range of 3-6 kDa, CHP binding should be possible. While we do not have specific validation data for this exact application, the binding mechanism of CHPs supports this. We encourage you to test it and would be interested in hearing about your results.

CHPs are versatile and have been successfully used on a wide variety of tissues. While our general user guides provide a strong starting point, minor optimization may be beneficial for unique tissues.

• Skeletal Muscle: Yes, CHPs can effectively stain skeletal muscle. For instance, B-CHP has been documented for staining the endomysium (Ref: Osokine et al., Aging Cell, 2023; https://onlinelibrary.wiley.com/doi/10.1111/acel.13936).
• Liver: CHPs are suitable for detecting collagen degradation in liver sections, including formalin-fixed paraffin-embedded (FFPE) tissues. We generally recommend B-CHP for its flexibility or our fluorescent CHPs (F-CHP, R-CHP). (Ref: e.g., "Damaged collagen detected by collagen hybridizing peptide as efficient diagnosis marker for early hepatic fibrosis").
• Brain Sections: Yes, CHPs can be applied to ex vivo brain sections to image changes in collagen, such as in the basement membrane of microvasculature (e.g., vessels <100µm diameter). While CHPs do not typically cross the intact blood-brain barrier (BBB) in live in vivo studies, this is not a constraint for tissue sections. Staining of basement membrane collagen is possible, though the signal intensity or consistency might be different compared to fibrillar collagens due to lower abundance or accessibility.
• Amnion Membrane: CHPs can be used to visualize collagen structure in amnion membranes, including those that have been crosslinked (e.g., with glutaraldehyde, though this may increase autofluorescence). Please refer to our general user guides on the Resources page of our website for starting protocols.

We are always happy to discuss specific modifications needed for your particular tissue type or experiment.

Our In vivo CHP, labeled with sulfo-Cyanine7.5 (sCy7.5), is specifically designed for live animal imaging. A key feature of this CHP is its modified sequence which allows it to be directly injected into animals without requiring a pre-activation heating step, unlike our other CHP formulations for ex vivo use.

Yes, our In vivo sCy7.5 CHP can be used for histology on paraffin-embedded or frozen tissue sections after in vivo experiments or independently. However, please note that it is more expensive than our CHPs specifically optimized for histology (F-CHP, R-CHP, B-CHP). If you choose to use the sCy7.5 CHP on tissue sections, we recommend an overnight incubation for optimal binding.

Tissue autofluorescence, particularly in the green channel, can sometimes interfere with F-CHP signals.

• Recommendation: Switching to our R-CHP (Cy3 conjugate, red fluorescence, Ex/Em ~550 nm / ~570 nm) often helps minimize this issue, as autofluorescence is typically lower in the red channel.
• Other strategies: Ensure proper fixation (avoid glutaraldehyde), consider using spectral unmixing if your imaging system supports it, or use appropriate image processing techniques. For B-CHP, choosing a far-red or near-infrared secondary fluorophore can also help.

Yes, we have an ImageJ/Fiji protocol detailed in our application notes (available on our Resources page) that can be adapted for quantifying CHP staining. This is applicable whether you are using B-CHP (with a secondary detection step resulting in fluorescence or chromogenic signal) or directly fluorescent CHPs (F-CHP, R-CHP). The general workflow involves:

1. Converting the image to an appropriate bit depth (e.g., 8-bit or 16-bit).
2. Appropriate background subtraction or correction.
3. Adjusting contrast and setting a signal threshold to define positive staining.
4. Measuring parameters like percentage area stained or mean fluorescence intensity within regions of interest.

This protocol provides a general framework and may need adjustment based on your specific image quality and experimental question.

We have not observed a significant change in CHP staining efficacy or signal intensity directly correlated with minor variations in cryosection thickness (e.g., 5 µm vs 10 µm). Standard section thickness appropriate for your tissue type, antibody penetration (if co-staining), and imaging setup (e.g., objective working distance) should be suitable. Very thick sections might experience more background due to light scattering or trapping of unbound peptide.

We may be able to accommodate requests for custom CHP synthesis, including different labels or specific chemical modifications (e.g., a free thiol group for conjugation). Please contact us directly at info@3helix.com with the details of your specific requirements:

• Desired sequence (if different from standard)
• Type of modification or conjugation
• Required quantity
• Intended application

Custom synthesis typically involves a longer lead time (e.g., estimated 10–12 weeks, but this can vary) and may require specific agreements regarding the peptide's use. We can provide a quote upon evaluation of your request.

We announced a CHP-based cosmetic ingredient in development in partnership with BASF in the fall of 2023. This product is currently still under development and not yet commercially available. Updates will be provided as they become available.

1. Converting the image to an appropriate bit depth (e.g., 8-bit or 16-bit).
2. Appropriate background subtraction or correction.
3. Adjusting contrast and setting a signal threshold to define positive staining.
4. Measuring parameters like percentage area stained or mean fluorescence intensity within regions of interest.

This protocol provides a general framework and may need adjustment based on your specific image quality and experimental question.

Our standard, commercially available CHPs (which are conjugated to dyes or biotin) are primarily designed for imaging and detection of denatured collagen, not for bulk collagen removal or depletion.

You can request a quote through our website or by contacting our sales/customer service team directly via email at orders@3helix.com or info@3helix.com.

We are happy to assist with this. We can provide a formal price quote, a proforma or sample invoice that includes our banking details, our W9 form (for US institutions), EIN, company address, and other necessary business information. For international customers, please let us know if a VAT number, company registration number, or any other specific documentation is required for your institution's supplier registration system. Please contact orders@3helix.com or info@3helix.com.

Currently, 3Helix does not offer samples. We understand that trying a product can be important, and while we don't have a sample program, we're proud that our products have been successfully utilized in hundreds of peer-reviewed publications and cited thousands of times. We'd be happy to discuss your specific research needs to see if CHPs are the right fit for you!"

We are always ready to help! Please reach out to our dedicated support team with any questions regarding product selection, protocol optimization, data interpretation, or troubleshooting. Our scientific staff has the expertise to assist you.

• info@3helix.com.
• 385-722-4772

Comprehensive user guides, protocols, and application notes for our various CHP products and applications are available on our website. You can typically find these on the individual product pages and on our dedicated Resources page. Downloadable PDF versions of the user guides are available there.

Currently, our primary user guides and technical documentation are provided in English. These are available online, and we can send PDF versions of these English guides via email. If you have a specific language requirement, please let us know. While we cannot guarantee immediate availability of translated documents, we can note the interest and explore possibilities if there is significant demand.

CHPs are shipped in powder form.

• Domestic (US): FedEx two-day service.
• International: FedEx Priority International service.

CHPs are very stable in powder form and can be shipped without temperature controls (e.g., no ice packs required). However, upon receipt, we recommend storing the peptide powder at -20°C for long-term storage.

• Domestic (US): Once your order has shipped, it should typically arrive in 2 business days.
• International: International shipments usually take about 3-5 business days, but this can vary depending on the destination and customs processing. You will receive an email with tracking information once your order has been processed and shipped.

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If you require ordering via Purchase Order, we accept payments on a Net 30 basis. You may check out on our website using a Purchase Order, or you may email your purchase orders to orders@3helix.com.

Due to the nature of the peptides we sell (research reagents, often requiring specific storage and handling), we generally do not accept returns. However, 3Helix stands behind the quality of our products. If you encounter a quality control issue or have concerns about the product's performance, please contact us immediately via email at orders@3helix.com or info@3helix.com so we can work with you to resolve any issues.

Yes! You can use your own FedEx account. Please provide 3Helix with your FedEx account number when placing your order, and we will waive our shipping fee and charge the shipping cost directly to your FedEx account.