3Helix CHP Application: Quantifying Collagen Content

Fluorescent quantification of total collagen

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Introduction

3Helix Collagen Hybridizing Peptides (CHPs) are short peptides that selectively target damaged and remodeling collagen for applications in therapeutics, cosmetics, drug delivery systems, and life science tools.

Summary

Collagen Quantification Application: Total collagen staining with CHPs; a step-by-step guide with an Image-J / FIJI imaging protocol.

Application Note

Total Collagen Content: To use CHPs for evaluating the total collagen content within an FFPE tissue section (after deparaffinization) or a frozen tissue section, the collagen must be fully denatured to allow for CHP binding on all available collagen. We recommend heating the tissue section to thermally denature the collagen. Our tests indicate that the long heating periods used in heat-induced epitope retrieval (HIER) methods are sufficient to completely denature the collagen in the sample (regardless of the buffer used). The tissue sections shown in this application note were placed in 50 mL of citrate buffer in a tissue steamer for 25-45 minutes at 95-100 °C. Alternatively, to denature the collagen content without a steamer, a water bath can be used to heat up a sealed 50 mL tube containing DI water to temperatures over 85 °C. After heating, pipette the hot DI water onto the tissue samples and allow to sit for 5 minutes (repeat 5X).

Figure 1.: Normal rabbit skin sections of different thickness stained with 20 um R-CHP after thermal antigen retrieval. As the thickness of the tissue sections increase, more CHP intensity is observed. Signal quantification using ImageJ/FIJI shows a linear correlation between tissue thickness and signal intensity.

Figure 1: Normal rabbit skin sections of different thickness stained with 20 um R-CHP after thermal antigen retrieval. As the thickness of the tissue sections increase, more CHP intensity is observed. Signal quantification using ImageJ/FIJI shows a linear correlation between tissue thickness and signal intensity.

Experimental Protocol

  1. FFPE Sections: perform deparaffinization prior to CHP staining by submerging sections for 2 x 5-minute washes in xylenes, 100% ethanol, 95% ethanol, 50% ethanol, and DI water in this order.
  2. Perform HIER or purposefully heat denature the tissue for the determination of total collagen content.
  3. Create a working solution for CHP staining by dissolving CHP powder in 1× PBS so that the concentration is within a 5-20 μM range. The exact concentration depends on the optimized parameters for the tissue section and the volume needed.
  4. Heat CHP solution to 80 °C. Since CHP can self-assemble into homotrimers in solution over time (e.g., during storage at 4 °C) and lose its driving force for collagen hybridization, the trimeric CHP needs to be thermally dissociated to single strands by heating briefly at 80°C.
  5. Quickly quench hot CHP solution in an ice water bath (~30 sec) before using it to bind to unfolded collagen in the tissue.
  6. Apply quenched CHP solution to tissue section. Completely cover tissue section with CHP staining solution and allow overnight binding at 4 °C in a humidity chamber.
  7. Wash sections using 3 x 5-minute washes of 1× PBS or 1× Tween-20.
  8. Mount coverslips and image.

Image-J/FIJI Macro Steps

Please follow the steps below to complete the image analysis correctly. The comments describe what each step of the macro code does, and they are shown in pink. We have also provided the code without comments so that you can copy and paste it directly into ImageJ by going to Plugins > New > Macro.

NOTE: The user will need to determine an acceptable threshold value to help remove some of the background auto-fluorescence. This macro works best with high signal-to-noise ratio, if there is low signal with lots of background noise or auto-fluorescence, you will need to include additional steps prior to running this macro.

setBatchMode(true); //Tells imageJ that the following code will be run on a batch of files.
output = getDirectory("Output Directory"); //sets the output variable as the user defined directory...
input = getDirectory("Input Directory"); //sets the input variable as the user defined directory...
Dialog.create("File Type");
Dialog.addString("File Suffix: ",".tif"); //tells ImageJ to only use files of type “.tif”...
suffix = Dialog.getString();
processFolder(input); //This is how ImageJ will search through the user specified input folder.
... and so on ...
This is the macro code without comments, ready to be copied directly into ImageJ:

setBatchMode(true);
output = getDirectory("Output Directory");
input = getDirectory("Input Directory");
Dialog.create("File Type");
Dialog.addString("File Suffix: ",".tif");
suffix = Dialog.getString();
processFolder(input);
... and so on ...

NOTE: This process was designed based of previous literature that focused on the ImageJ/FIJI image quantification tools and methods.4–6 We want to provide the most effective and efficient protocol for our customers. As such, we ask that if you have optimized these steps or found an alternative tool within ImageJ/FIJI that works better, please contact us and we will update this application note and acknowledge your contribution!

How Does CHP Staining Compare to Other Methods?

CHP staining of fibrotic liver tissue performed just as well if not better than other common stains for detecting collagen in tissue sections. Expert pathologists noted that CHP staining “performed better for collagen detection [than other stains], seemed to have a consistent staining pattern, and was easy to interpret.” In automated image analysis, CHP staining quantified collagen area as accurately as PSR, and more accurately than Masson’s Trichrome, and Herovici’s stain. Overall, CHP is a one-step collagen stain that has high contrast and is easier to read than other collagen histology stains, while maintaining high accuracy.

 

Ilex Life Sciences LLC is an authorized distributor of CHP products manufactured by 3Helix, Inc.