Assessing collagen deposition during aging in mammalian tissue and in Caenorhabditis elegans

Abstract

Proper collagen homeostasis is essential for development and aging of any multicellular organism. During aging, two extreme scenarios are commonly occurring: a local excess in collagen deposition, for instance during fibrosis, or a gradual overall reduction of collagen mass. Here, we describe a histological and a colorimetric method to assess collagen levels in mammalian tissues and in the nematode Caenorhabditis elegans. The first method is the polychrome Herovici staining to distinguish between young and mature collagen ratios. The second method is based on hydroxyproline measurements to estimate collagen protein levels. In addition, we show how to decellularize the multicellular organism C. elegans in order to harvest its cuticle, one of two major extracellular matrices, mainly composed of collagen. These methods allow assessing collagen deposition during aging either in tissues or in whole organisms.

1. Introduction

Cells and tissues are embedded within extracellular matrices (ECM), which are important for tissue geometry, integrity, and function (1). In mammals, collagens constitute about 30% of the total protein mass in the body and are the major component of the ECM (2). In humans, collagen turnover can be extremely slow as observed in the lens of the eye and in cartilage structures (with a half-life time of 114 years (3–5)) or extremely fast, within 72 hours after physical exercise, in Achilles tendons (6). Hence, depending on the tissue or organ, some collagens are synthesized, secreted, and integrated in the ECM once early in life and are not replaced over the entire lifetime, whereas other collagens have a higher turnover rate.

During aging, the connective tissue or ECM integrity declines due to accumulation of damage from collagen fragmentation, oxidation, and glycation (2, 7–12) leading to a continuous reduction of collagen mass, as best illustrated by wrinkles and sagging skin (13, 14), but also observed in other connective tissues that support organ function. This deterioration in ECM integrity has been implicated in many age-dependent diseases, such as diabetes, cancer, chronic liver diseases, and cardiovascular diseases (2, 7–12). In parallel, the accumulation of molecular damage (15), chronic inflammation (inflammaging) (16), or injury during aging might locally drive abnormal collagen deposition resulting in fibrosis (17). These observations suggest that in general the overall collagen mass tends to decline as a function of age, but due to the increasing incidence of damage or injury, atypical collagen accumulation might occur more locally in certain parts of the body. Therefore, proper assessment of the extent of collagen deposition might reveal novel insights in maintenance and remodelling of the ECM during aging.

A direct role for proper collagen deposition and remodelling during aging has become evident by studying model organisms, such as mice and C. elegans. For instance, deficits in the ECM remodelling enzyme (MMP14) leads to premature aging, short lifespan, and cell senescence in mice (18). Interestingly, long-lived mice preserve connective tissue elasticity and integrity during aging (19, 20). Consistent with these findings, in the nematode C. elegans, collagen synthesis declines during aging. Moreover, most if not all long-lived C. elegans mutants delay this progressive decrease in collagen mass (21). Delaying this progressive reduction in collagen mass by actively prolonging the biosynthesis of collagens during lifetime is required and sufficient for healthy aging and longevity in C. elegans (21).

In addition to studying the progressive age-dependent decrease in ECM integrity in mice, C. elegans provides additional unique opportunities to explore this phenomenon during aging, since: 1) C. elegans is transparent thereby allowing ECM components to be tagged by fluorescent proteins to directly monitor ECM homeostasis and integrity non-invasively in vivo (21); and 2) C. elegans is a well-established aging model because of its short lifespan (about 3 weeks) and powerful established genetics. However, histological methods are not as commonly used or even developed in C. elegans compared to mice. Below, we describe a histological method (Herovici’s staining) to be used for both C. elegans and mammalian tissue, a colorimetric method (measurement of hydroxyproline levels) to estimate collagen levels in both C. elegans and mammalian tissue, and a technique to isolate the C. elegans cuticle (extracellular matrix) to be used for further assessment of collagen deposition or other analysis.

The Herovici staining protocol (22) is one of several histological methods used to assess the extent of collagen deposition in a tissue. Compared to other widely used collagen staining techniques (e.g., picrosirius red staining or Masson’s trichrome staining), the Herovici protocol allows to distinguish between young (e.g., collagen III) and mature collagen (e.g., collagen I) without cross polarized filters, providing an obvious advantage in the visualization and analysis of processes, where changes in collagen ratios are expected or known to occur. The sequential use of several dyes results in a polychromatic staining, with young collagen appearing blue, mature collagen colored pink to red and cytoplasm counterstained in yellow (Fig. 1). Moreover, cell nuclei are stained dark blue to black by Haematoxylin. The Herovici method is mostly used for histological studies in skin, however, the staining protocol works well in other tissues or organs too (e.g., liver or lung).

Fig. 1. Herovici’s staining for mammalian tissue and C. elegans.

(a-c) A schematic of the individual steps of the Herovici’s staining described in the method section (see Method 3.1). (a) Freeze-cracking of C. elegans (see Method 3.1.1). (b) Mammalian tissue harvesting and deparaffinization (see Method 3.1.2). (c) Shared part of the Herovici’s staining protocol for both C. elegans and mammalian tissue (see Method 3.1.3). (d) Herovici-stained mouse skin specimen. The young collagen (collagen type III) is stained in blue, whereas the mature collagen (collagen type I) is stained in red. Cytoplasm is counterstained in yellow, cell nuclei dark blue to black. Three layers forming the skin (epidermis, dermis, hypodermis) can be identified. (e) Herovici-stained C. elegans at day 1 of adulthood.

Collagen levels can also be determined by using hydroxyproline measurements. Collagens have characteristic [Gly-X-Y] repeats, whereby glycine is at every third position, X is frequently proline, and Y is frequently 4-hydroxyproline. These prolonged stretches of [Gly-X-Y] repeats in the collagen protein are important to form a stable triple helix from the three constitutive collagen chains (2). Since collagens are enriched in hydroxyproline and this posttranslational modification is not as common as in other proteins, measuring hydroxyproline abundancy results in an estimate of total collagen levels (23, 24). For a more accurate estimation of collagen amounts by hydroxyproline measurements, one could decellularize tissue samples of interest (25) in order to decrease the levels of other hydroxyproline containing proteins found in the cytoplasm.

Here we show techniques to isolate the C. elegans cuticle, a procedure to further enrich for collagen content during sample harvesting for instance for hydroxyproline measurements. The cuticle and basement membranes are the two major extracellular matrices of C. elegans (26). The C. elegans cuticle is mainly composed of collagens (27). Two methods are described to isolate this cuticle. The first and simpler method was modified from Leushner et al., 1979 (28) and the second method was adapted from Cox et al., 1981 (29). The advantages and differences of each method are explained in the method section below.

In summary, collagen deposition and remodelling become faulty during aging resulting either in a generalized decline in collagen biosynthesis or, locally, in excessive and amorphous collagen deposition (fibrosis). The methods described here are tailored for mammalian tissue samples and the organism C. elegans to enable the quantification of total collagen protein levels (hydroxyproline measurement), to distinguish between young and mature collagen ratios, and to enrich for collagen-containing extracellular matrices in C. elegans. In addition, we describe in detail how to synchronize C. elegans in order to start a time course for aging studies. With these methods in hand, the investigator should be able to assess collagen deposition during aging and in age-related pathologies.

2. Materials

General mouse necropsy and mammalian tissue harvesting or C. elegans maintenance are described elsewhere (30, 31). C. elegans strains can be acquired from the Caenorhabditis Genetics Center (CGC, https://cgc.umn.edu, (31)). For the preparation of all solution distilled water and analytical grade reagents are recommended. All Herovici’s staining solutions are stored in glass bottles at room temperature protected from light. Disposal of waste materials should be carried out according to specific local regulations. Suitable protection (lab coat, safety glasses, and gloves) and the use of a fume hood are recommended for the handling of acids and other hazardous substances.

2.1. Herovici’s staining for mammalian tissue and C. elegans

1. 70 - 100% EtOH.

2. Microscope slides with cut edges and frosted end, 1mm thick, 76x26mm.

3. Poly-L-Solution: 400 mg Sigma P1524 poly-L-Lysine; 0.2 g Sodium azide (0.1%) in 200 ml ddH₂O. Can be re-used and stored at 4°C.

4. PBS: 8 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, 0.24 g KH₂PO₄, pH adjusted with HCl to pH 7.4).

5. PFA: 4 % Paraformaldehyde solution in PBS.

6. Herovici’s Solution 1: Add 270 ml of deionized water to a glass beaker. Weigh 1.25 g Celestine Blue powder and transfer it to the glass beaker. Weigh 15 g of Aluminum potassium sulfate dodecahydrate (CAS: 7784-24-9; 31242, Merck) and add it to the glass beaker. Heat and boil the solution until the powders are dissolved (approximately 3 – 5 minutes). Let the solution cool down to room temperature and transfer to a 500 ml graduated cylinder and add distilled water to a final volume of 300 ml. Add 60 ml of glycerol once the solution has cooled down to room temperature (see Note 1).

7. Herovici’s Solution 2: Weigh 1 g of haematoxylin powder, transfer it to a glass beaker and dissolve it in 100 ml of 95% ethanol in distilled water (alcoholic haematoxylin). Weigh 4.5 g of ferrous II sulphate (FeSO₄) powder and 2.5 g ferric III chloride (FeCl₃) powder and transfer them to a separate glass beaker containing 298 ml distilled water. Add 2 ml of 36% (concentrated) hydrochloric acid (toxic, see Note 2). Stir the solution on a magnetic stirrer till powders dissolve (see Note 3). Mix this solution with the alcoholic haematoxylin from step 1 (see Notes 4, 5, and 6).

8. Herovici’s Solution 3: Add 300 ml of distilled water to a glass beaker. Weigh 1.25 g of metanil yellow and transfer it t the glass beaker. Add 25 drops (use a 1 ml pipette tip) of glacial acetic acid (see Notes 7 and 8).

9. Herovici’s Solution 4: Add 1800 ml of distilled water to a glass beaker. Add 9 ml of glacial acetic acid (toxic, see Notes 7 and 9).

10. Herovici’s Solution 5: Weigh > 1 g of lithium carbonate (Li₂CO₃) powder and dissolve it in 78 ml distilled water (aqueos Li₂CO₃). The solution will be saturated (see Note 10). Add 1 ml of saturated aqueous Li₂CO₃ to 500 ml distilled water to a glass beaker (see Notes 11 and 12).

11. Herovici’s Solution 6: Weigh 0.15 g of methyl blue powder and dissolve it in 150 ml distilled water in a glass beaker. Weigh 0.2 g of acid fuchsin powder and dissolve it in 150 ml of saturated aqueous picric acid in a separate glass beaker. Mix the two solutions together. Add 30 ml of glycerol and 0.15 ml of saturated aqueous Li₂CO₃ (see Notes 11 and 13).

12. Herovici’s Solution 7: Add 10 ml of glacial acetic acid to 1 L of distilled water (see Notes 7 and 14).

2.2. Total collagen quantification for mammalian tissue and C. elegans

2.2.1. Age-synchronization of C. elegans

1. 5 N NaOH.

2. 5% sodium hypochlorite solution (or household bleach).

3. Sterile ddH2O.

4. M9 buffer: 3 g KH₂PO₄, 7.52 g Na₂HPO₄, 5 g NaCl and 0.05 g MgSO4 in 1 L ddH2O, autoclaved (31).

2.2.2. Total protein quantification

1. QuickZyme Protein Assay Kit (QuickZyme Biosciences) or equivalent kit from other manufactures suitable for protein quantification of acid hydrolyzed samples.

2. Pierce™ BCA Protein Assay Kit (Thermo Fisher) or equivalent kit from other manufactures.

2.2.3. Total collagen quantification

1. QuickZyme Total Collagen Kit (QuickZyme Biosciences) or equivalent kit from other manufactures.

2. Aqueous hydrogen chloride solution: 4 M, 6 M, and 12 M.

2.3. Materials for cuticle (extracellular matrix) isolation in C. elegans

2.3.1. Cuticle isolation (freeze-thaw protocol)

1. Sodium dodecyl sulfate (SDS) buffer: 1% SDS in ddH₂O.

2. Triton X-100 buffer: 0.5% Triton X-100 in ddH₂O.

3. 15 ml glass conical centrifuge tubes with screw gaps.

2.3.2. Cuticle isolation (ST buffer protocol)

1. M9 buffer: 3 g KH₂PO₄, 7.52 g Na₂HPO₄, 5 g NaCl, 0.0493 g MgSO₄ in 1 L ddH₂O, autoclaved.

2. Sonication buffer: 10 mM Tris-HCl, pH 7.4; 1 mM EDTA; in ddH₂O.

3. 0.1 M PMSF buffer: 1 mM phenylmethanesulfonylfluoride in isopropanol

4. ST buffer: 1% SDS; 0.125 M Tris-HCl, pH 6.8; in ddH₂O.

3. Methods

3.1. Herovici’s staining for mammalian tissue and C. elegans

All procedures are carried out at room temperature, if not indicated otherwise. Glass slides with tissue sections fixed in 4% paraformaldehyde (PFA in PBS) are used for the Herovici staining protocol. Reusable Herovici solutions can be poured back in their original container. At the end of this procedure tissue sections will be ready to be imaged under the microscope. In this section, we first describe the harvesting and preparation for mammalian tissue (see Method 3.1.1) and then for C. elegans (see Method 3.1.2) followed by the shared methodical part (see Method 3.1.4).

3.1.1. Harvesting and preparing mammalian tissue sections (Fig. 1a)

1. Organs or tissues of interest are harvested as described elsewhere (30), fixed in 4% paraformaldehyde for 24 hours at 4°C, submersed in increasing concentrations of ethanol, embedded into paraffin (32), and sectioned with a microtome (3.5 µm sections).

2. Deparaffinize tissue sections by immerging the glass slides in xylol for 10 minutes. Repeat this step once for a total of two.

3. Rehydrate the tissue sections stepwise from 100% to 50% (100%, 96%, 90%, 80%, 70%, 50%) ethanol by immerging the glass slides for 1 minute in each solution.

4. Continue the protocol see section 3.1.4.

3.1.2. Preparation of Poly-L-Slides for freeze-cracking of C. elegans

1. Clean microscope slides with Kimwipes and 70%-100% EtOH.

2. Pipette 50 µl Poly-L-Solution on the non-frosted part of the microscope slide. Place another microscope slide on top, so that the transparent parts completely overlay and the frosted parts are hanging over on the opposite sides.

3. Slide the two slides apart: one side per slide should now be covered with a thin layer of Poly-L-Solution.

4. Turn the slides around, so that the Poly-L-Solution sides are facing upwards and place the slides in a 55°C incubator for 1 hour to dry. Slides can now be used directly or stored for several months in a clean slide box at 4°C.

3.1.3. Harvesting and preparing C. elegans samples using a freeze-cracking method (Fig. 1b)

The described C. elegans freeze-cracking method was adopted from Duerr 2006 (33). For freeze-cracking use a minimum of 4’000 C. elegans or better about 8’000 - 12’000 C. elegans (see Note 15). For a protocol to age-synchronize and culture C. elegans (see Method 3.2.2)

1. Place the fixation solution (4% paraformaldehyde (PFA in PBS)) and several empty 10 cm petri dishes on ice in order to pre-chill for later use.

2. Wash C. elegans off culturing plates using M9 buffer and collect them in a 15 ml conical centrifuge tube.

3. Centrifuge for 1 minute at 1000 rpm (standard table centrifuge is ca. 200 g; see Note 16) and remove supernatant. Wash the C. elegans until the supernatant is clean and no bacteria are visible (see Note 17).

4. Rinse C. elegans by filling up the 15 ml conical centrifuge tube with 14 ml ddH₂O, centrifuge for 1 minute at 1000 rpm (200 g), remove supernatant and add ddH₂O to the pelleted C. elegans to a total volume of 100 µl.

5. Using a glass Pasteur pipette, place a small drop of animals (ca.10 - 20 µl) on a Poly-L-Solution covered slide. Wait shortly to let the C. elegans settle on the slide. Use the pipette to spread the C. elegans out so that they do not overlap.

6. Carefully place another Poly-L-Solution covered slide on top of the slide with the C. elegans so that the Poly-L-covered sides face each other, while the frosted parts are not overlapping.

7. Put on safety goggles and gloves. Hold the two slides slightly pressed together into liquid nitrogen until the bubbling stops. Take the slides out. Wait for ca. 20 seconds. Then start to snip with your finger carefully against one of the slides until they rip apart.

8. Check which slide has most of the C. elegans attached to it and place either only this slide or both slides separately into individual empty 10 cm petri dishes, which are standing on ice.

9. Pipette 100 - 200 µl 4% PFA pre-chilled onto the C. elegans covered part of the slides. Fix the C. elegans by incubating the slides at 4°C for at least 30 minutes or up to 24 hours.

10. After fixation, rinse the slides gently at least three times with PBS. Proceed immediately with staining or store the slides in a coplin jar or petri dish containing PBS at 4°C for several days.

3.1.4. The Herovici staining for both mammalian tissue and C. elegans (Fig. 1c)

1. Immerse slides in Solution 1 for 5 minutes (see Note 1).

2. Rinse slides under running tap water for 2 minutes (see Note 18).

3. Immerse slides in Solution 2 for 5 to 6 minutes (see Notes 5, 6, and 19).

4. Rinse slides under running tap water for 2 minutes (see Note 18).

5. Immerse slides in Solution 3 for 2 minutes (see Note 8).

6. Transfer slides directly to Solution 4 for 2 minutes (see Note 9).

7. Rinse slides under running tap water for 2 minutes (see Note 18).

8. Immerse slides in Solution 5 for 2 minutes (see Note 12).

9. Transfer slides directly to Solution 6 for 2 minutes (see Note 13).

10. Transfer slides directly to Solution 7 for 2 minutes (see Note 14).

11. Immerse the slides in 100% ethanol for 1 minute. Repeat this dehydration step once for a total of two.

12. Immerse the slides in xylol for 2 minutes. Repeat this step once for a total of two.

13. Let the slides dry for a minimum of 10 minutes (see Note 20).

14. Mount the slides with a mounting media (e.g. Eukitt®) and let them dry under a fume hood (see Note 21).

15. After adding the cover slip, the specimen is ready for imaging. Examples of a Herovici stained mammalian tissue section and a C. elegans are shown in Fig. 1d and Fig. 1e, respectively.

3.2. Total collagen quantification for mammalian tissue and C. elegans

The collagen content in a tissue or whole-organism sample can be determined in multiple ways (Fig. 2). It is generally advised to select the normalization entity to be as conclusive as possible. Here, total collagen quantification is combined with either absolute animal number (C. elegans) or with total protein determination for both mammalian tissue and C. elegans. Here, we focus mainly on how to establish aged C. elegans cultures and on the assessment of collagen levels in C. elegans, since the use of hydroxyproline measurement in mammalian tissue is well-established.

Fig. 2. Total collagen quantification using hydroxyproline assay for mammalian tissue and C. elegans.

(a-b) Schematic of the collagen assay that can be performed using mammalian tissue (a) as well as C. elegans samples (b). Please, see method section (see Method 3.2) for detailed protocol steps. (c) An example of how to quantify the collagen to protein ratio for larval L4 stage spe-9(hc88) C. elegans samples are shown. (d) Formula (1) shows how to calculate the ratio of collagen content to total protein levels. The dilution term referring to the digestion is omitted when the total protein content is determined using the hydrolyzed sample. Formula (2) shows how to normalize collagen content per individual C. elegans.

3.2.1. Mammalian tissue (Fig. 2a)

There are several ways to assess hydroxyproline levels in mammalian tissues. Here, we briefly outline the use of a colorimetric method using the QuickZyme kit for both hydroxyproline and total protein measurements. The advantage of this protocol is that each HCl hydrolyzed tissues sample can be split to measure 1) hydroxyproline levels to estimate collagen protein content and 2) to measure total protein levels from the same sample in order to normalize the collagen content to total protein levels. Equivalent kits or protocols from other manufacturers can be used. For more details, please follow the manufacturer's instruction.

1. Organs or tissues of interest are harvested as described elsewhere (30). Frozen organs/tissues, formalin-fixed or paraffin embedded tissues can also be used.

2. Hydrolyze the tissue in 6 M HCl in a safety centrifuge tube for 16 hours at 95°C in a one 1:10 ratio of wet tissue weight to acid volume (see Note 22). Split the hydrolysate: 35 μl is used for collagen quantification and 15 μl to assess the total protein content (see Note 23).

3. To generate the standard curve for the colorimetric quantification of collagen and total protein levels, please follow the manufacturer’s instructions.

3.2.2. Synchronizing C. elegans for an age-dependent time course analysis (Fig. 2b)

C. elegans samples are prepared in one batch and harvested in user defined intervals over the animal’s lifespan. To age-synchronize the C. elegans population, we describe a bleach preparation protocol to harvest eggs from gravid C. elegans adults. For each time-point and condition 2’000 - 5’000 animals are required, therefore ca. 10’000 - 100’000 eggs should be harvested in the initial lysis step.

1. Use multiple culturing plates containing a large amount of gravid C. elegans adults to perform a population lysis. Wash the culturing plates with sterile H₂O. Pipet the H₂O across the plate several times to loosen C. elegans that are stuck onto the bacterial lawn.

2. Collect the liquid containing the C. elegans in a sterile 15 ml conical centrifuge tube. Centrifuge for 1 minute at 1000 rpm (standard table centrifuge is ca. 200 g; see Note 24). Discard supernatant. Wash residual bacteria away from C. elegans by filling up the 15 ml conical centrifuge tube with sterile H₂O, spin down and discard the supernatant. Repeat this step until the supernatant is clear. Spin down to pellet gravid C. elegans and add H₂O to a total volume of 3.5 ml.

3. Put on gloves and eye protection. Add 1 ml of 5% sodium hypochlorite solution (or household bleach) and add 0.5 ml of 5N NaOH. Close 15 ml conical centrifuge tube and shake well. In one-minute intervals, hold the 15 ml conical centrifuge tube under a dissecting scope to observe lysis of gravid C. elegans and shake well after observation to optimize lysis. Once almost all C. elegans are lysed add M9 buffer to a total volume of 15 ml and centrifuge at 3000 rpm (ca. 1800 g) for 1 minute to pellet the eggs (see Note 25). Discard supernatant and wash the eggs at least three times by filling up to 15 mL with M9 buffer and re-pelleting the eggs by centrifugation at 3000 rpm (ca. 1800 g) for 1 minute. Discard the supernatant (see Note 26).

4. After lysis and subsequent washing steps the samples are incubated while rotating for 12 h in M9 buffer supplemented with 5μg/ml cholesterol.

5. 500 - 1000 larval L1 C. elegans larvae are placed on each 10 cm culturing plate. If a temperature-sensitive sterile strain is used the sterility mechanism has to be activated at the needed time point (see Note 27). If floxuridine (FUDR) is used to avoid offsprings, the animals are transferred at the larval L4 stage to 50 μg/ml FUDR culturing plates (see Note 28 and 29).

6. 2000 - 5000 C. elegans (2 - 10 plates) are harvested for each condition and time point. Transfer all animals from the plates to a 15 ml conical centrifuge tube. The animals are centrifuged briefly at low speed (200 rpm, ca. 8 g) to pellet aged C. elegans adults. The suspension containing unhatched eggs or larvae (if FUDR is used) is aspirated and discarded. Repeat this washing step 3 times or until only the desired C. elegans adults are present and supernatant is clear (see Note 30).

7. From the last wash that was filled up with M9 buffer to a total volume of 14 ml take five times 20 µl aliquots and place these aliquots on a petri dish lid to count the number of animals in each 20 µl drop using a dissecting scope (see Note 31 and 32). To estimate total number of C. elegans, average the number of C. elegans from each 20 µl drop and multiply by 700 to get an estimate of the total number of C. elegans present in 14 ml sample.

8. Centrifuge for 1 minute at 1000 rpm (ca. 200 g) to softly pellet C. elegans. Discard supernatant. Use a glass Pasteur pipette to transfer the C. elegans pellet to a labelled 1.5 ml Eppendorf collection tube, fill up with sterile H₂O to a total volume of 200 µl and store at -80°C (see Note 16, 33, and 34).

9. At the end of the time course, once all samples have been collected, proceed by thawing all tubes. At least 3-5 freeze-thaw cycles should be performed by thawing at room temperature and freezing in liquid nitrogen (or dry ice with EtOH, or by placing back into the -80°C).

10. Then sonicate all samples on ice until all animals are disrupted. The C. elegans cuticle is the hardest to break. With a standard sonication device, it will take several repeats to break down all C. elegans (see Note 35).

3.2.3. Total protein quantification for C. elegans (Fig. 2)

To determine the total collagen content as a fraction of total protein abundance, the total protein content has to be determined separately.

1. Pipette 25 µl of each sonicated sample as well as of the provided standard solutions into a 96-well plate and quantify the total protein content following the manufacturer’s instructions (Pierce™ BCA Protein Assay Kit, Thermo Fisher or equivalent kit from other manufacturers). If needed, dilute the sample with M9.

2. Use a plate reader to quantify the color change at the specified wavelength.

3.2.4. Total collagen quantification for C. elegans (Fig. 2)

Here, we determine the total collagen abundance by digesting the sample and directly measuring the concentration of the amino acid hydroxyproline in each sample.

1. The sonicated samples are transferred into heat-stable tubes provided by the kit manufacturer (QuickZyme Total Collagen Kit, QuickZyme Biosciences or equivalent kit from other manufacturers). The provided collagen standard does not require to be sonicated but is otherwise treated identically as the samples.

2. According to the manufacturer’s instructions the sample and standard are mixed with 12M HCl solution and incubated for 20 hours at 95°C.

3. In brief, after the digestion the supernatant is isolated, the acid content lowered, assay buffer added and the plate incubated at room temperature for 20 minutes followed by the addition of the detection reagent and subsequent incubation at 60°C for 1 hour. The color change is quantified using a plate reader (see Note 36).

4. The total collagen content per sample in a well can be directly determined through the measured color change. In a first step, the collagen reference sample (1.2 mg / ml of rat tail collagen) is used to generate a standard curve relating the magnitude of the color change to the collagen content of the well. Over the used concentration range this relationship is approximately linear. With the standard curve available, the collagen content of the samples can be quantified. It must be noted that the collagen standard supplied in the QuickZyme Biosciences kit contains rat tail collagen. To validate the use of rat tail collagen as a reference for hydroxyproline levels for C. elegans collagens, the potential hydroxyproline occurrences on the Y position in the C. elegans [Gly-X-Y] triple repeats in collagens were estimated in silico. The relative proline abundance on the Y position is similar between C. elegans and rat tail collagens (personal communication Jan M. Gebauer). Example results for C. elegans protein and collagen concentration are shown in Fig. 2b.

5. The total collagen to total protein ratio can be determined by combining the measured collagen and protein concentrations according to formula (1). In the case of C. elegans, the total collagen abundance per animal can be calculated by subjecting the measured collagen concentration to formula (2). All formulas to determine the relative collagen contents are depicted in Fig. 2c.

3.3. Methods for cuticle (extracellular matrix) isolation in C. elegans

3.3.1. Cuticle Isolation (freeze-thaw protocol)

This method was modified from Leushner et al., 1979 (28) for the use in C. elegans. The two advantages of using this protocol compared to the ST protocol is 1) its simplicity and 2) the SDS-cleaned cuticles might still contain Schiff base products or other adducts that might be of importance of the desired analysis (34). The major problem with this protocol is that the C. elegans cuticle tends to stick on plastic; this can be avoided by using glass materials instead. All steps after the freeze-thawing should be carried out on ice, except steps involving SDS. We recommend to use about 12’000 C. elegans per sample to isolate a sufficient number of cuticles. Please, see Methods 3.2.2 for a protocol to age synchronize the C. elegans samples. An example of an aged C. elegans (7-days adult) is shown in Fig. 3a for comparison with the isolated cuticles Fig. 3b and 3c.

Fig. 3. Isolated C. elegans cuticles.

(a) Differential contrast image of a day 7 adult C. elegans. Anterior to the left and vertral side down. (b) Intact C. elegans cuticle after 1% SDS before washing step. Black arrows point to the Annuli structure of the cuticle. White arrowheads point to debris. (c) Intact C. elegans cuticle after washing. Sometimes other extracellular matrices, such as the basement membranes of the gonad (white arrowheads) are still attached.

1. Use 12’000 adult C. elegans (about 8-12 full culturing plates). Pipette around 3 ml M9 buffer on each plate. Gently tilt plates to collect the liquid on one side and carefully pipette the M9 buffer several times across the plate to loosen up the C. elegans stuck on the bacterial food lawn. Transfer the animals from the plates into a 15 ml conical centrifuge tube.

2. Wash the C. elegans three times with 15 ml ddH₂O by centrifugation at around 1000 rpm (ca. 200 g) for 1 minute and discard the supernatant (see Note 33).

3. After the last washing step discard the supernatant again and use a glass Pasteur pipette to transfer the C. elegans pellet into a 1.5 ml Eppendorf microfuge tube (see Note 37). Centrifuge 1.5 ml tube at around 100 g for 30 seconds, discard supernatant and fill up with ddH₂O to 100 μl.

4. Freeze the tubes in liquid nitrogen for one minute (or until frozen), followed by a thawing step at room temperature. Repeat this freeze-thaw cycle at least three times.

5. Sonicate the samples five times in intervals of 20 seconds each at an amplitude of 80% with 20 seconds breaks on ice in between (Sonoplus mini20 from Bandelin) (see Note 38).

6. Use a glass Pasteur pipette to transfer the ruptured C. elegans to 15 ml glass tubes (with screw gaps) (see Note 39) and suspend them in 10 ml 1% SDS solution, followed by overnight incubation onto a rotor at 37°C. The 1% SDS should wash out all the internal cells and organs from the disrupted cuticles (Fig. 3b).

7. On the following day, wash the cuticles with sterile ddH₂O containing 0.5% Triton-X by centrifuging at 3000 rpm (ca. 1800 g) for 1 minute, discard supernatant (see Note 40).

8. The cleaned cuticles (Fig. 3c) can now be further processed, analyzed, or stored at -20°C.

3.3.2. Cuticle Isolation (ST buffer protocol)

This method was adapted from Cox et al., 1981 (29). Please, see Methods 3.3.1 for the comparison with the other cuticle isolation freeze-thaw method and see Methods 3.2.2 for a protocol to age synchronize the C. elegans samples. All steps after the sonication should be carried out on ice, except steps involving SDS.

1. Collect 12’000 (adult) C. elegans with M9 buffer in a 15 ml tube as described in the first step of Methods 3.3.1.

2. Wash the C. elegans pellet three times by centrifugation at around 1000 rpm (ca. 200 g) for 1 minute and discard the supernatant (see Note 33).

3. After the last washing step discard the supernatant and re-suspend the C. elegans pellet in 5 ml sonication buffer. Incubate on ice for 10 minutes.

4. Add 30 µl 0.1M PMSF to the solution and sonicate five times for 20 seconds with an amplitude of 80% with 20 seconds breaks on ice in between (Sonoplus mini20 from Bandelin).

5. Wash the ruptured C. elegans cuticles three times with the sonication buffer by centrifugation at 1500 rpm (around 450 g) for 1 minute and discard the supernatant.

6. Discard the supernatant and transfer the cuticles with a glass Pasteur pipette to a 1.5ml microfuge tube (see Note 37). Centrifuge the 1.5 ml tube at around 100 g for 30 seconds, discard supernatant and fill up with sonication buffer to 100 μl.

7. Add 1 ml ST buffer and heat the tube for 2.5 minutes at 95°C.

8. Incubate the tubes on a rotator overnight at room temperature.

9. Wash cuticles three times with 0.5% Triton-X in sterile ddH₂O by centrifuging at 3000 rpm (ca. 1800 g) for 1 minute; discard supernatant.

10. The cleaned cuticles can now be further processed, analyzed, or stored at -20°C.

4. Notes

4.1. Herovici’s staining for mammalian tissue and C. elegans

• 1. Solution 1 can be reused.

• 2. Caution. Hydrochloric acid should be handled with care and added to the solution under a fume hood.

• 3. If required filter the solution to eliminate visible impurities.

• 4. Alcoholic haematoxylin and the ferrous solution are prepared separately and mixed at the end.

• 5. Solution 2 has a shelf life of approximately 2-3 months due to oxidation.

• 6. Solution 2 can be reused.

• 7. Caution. Glacial acetic acid should be handled with care and added to the solution under a fume hood.

• 8. Solution 3 can be reused.

• 9. Do not reuse solution 4.

• 10. By adding more than 1 g of Li₂CO₃ to 78 ml distilled water, the solution will be saturated. Do not shake to try to dissolve the precipitate. Let the solution stand.

• 11. Pipette the aqueous Li₂CO₃ from the top without disturbing the precipitate accumulated on the bottom of the container.

• 12. Solution 5 can be reused.

• 13. Solution 6 can be reused.

• 14. Do not reuse solution 7.

• 15. Per plate you can make 2-3 freeze cracked C. elegans slides.

• 16. Bacteria prevent the animals to properly stick to the slides.

• 17. To prevent adhesion of animals to plastic pipette tips one can either add 0.5% Triton-X as a detergent or switch to glass pipettes.

• 18. Regular water. This step can be performed directly in the sink.

• 19. A longer incubation may be needed if the solution has not been freshly prepared.

• 20. A longer drying time (30 minutes – 1 hour) is perfectly fine.

• 21. Eukitt® will need approximately 30 minutes to dry.

4.2. Total collagen quantification for mammalian tissue and C. elegans

• 22. Ideally, a 1:10 ratio of wet tissue weight to acid volume is recommended, with a minimal volume of 200 μl 6 M HCl per tube. For example, 50 mg of tissue mixed with 500 μl of 6 M HCl can be used.

• 23. To be able to relate the total collagen content of the sample as robustly as possible to the total protein abundance we recommend to measure the later directly in the hydrolyzed sample. To be able to quantify the free amino acid level present in the acidic hydrolyzed sample a suitable kit has to be chosen. We recommend the Total Protein Assay, QuickZyme Biosciences.

• 24. When centrifuging C. elegans either turn off the breaks or turn down the deceleration to a minimum.

• 25. In general,omplete lysis can take about 4-8 minutes. Hence, it is important to regularly check if all C. elegans are lysed. If lysis is performed for too long, the eggs will suffer from increasing damage.

• 26. Sometimes three washes are not enough to wash out all bleach solution. Hence, check by smelling the 15 ml conical centrifuge tube if bleach odor is still present.

• 27. We recommend the C. elegans mutant spe-9(hc88). Due to a temperature-sensitive defect in spermatogenesis, the animals are not able to lay fertilized eggs when shifted to 25°C during development. To ensure all animals are sterile, L1 animals can be cultured at 25°C until day 1 or 2 of adulthood.

• 28. To avoid food scarcity check the grow-up plates often and if needed transfer to fresh plates.

• 29. To check the effectiveness of the intervention a selected number of plates can be used for validation by for example placing a fluorescent reporter strain on them.

• 30. Since the collagen content is changing over the lifespan of C. elegans it is crucial to work with age-synchronous populations. Thus, all eggs and larvae must be removed as best as possible. This can be achieved by transferring animals often to new plates, rigorous washing or through filtration.

• 31. Because adult C. elegans will sink to bottom of the conical centrifuge tube, shake the 15 ml conical centrifuge tube well between taking the aliquots for counting. Try to pipette from the middle of the 15 ml conical centrifuge tube for consistency.

• 32. Use a pipette with a large orifice to sample C. elegans during the counting procedure. Since adults grow in size a too small pipette tip can lead to an underestimation of older populations.

• 33. At this step of the protocol is a good time point for a break or to wait until all time points from before are collected. For instance, if you collect daily samples over the 2-3 weeks lifespan of C. elegans.

• 34. It is recommended to fill up all sample C. elegans pellets to 200 μl to simplify the downstream steps.

• 35. Example sonication configuration using a Bandelin Sonoplus, UW mini20 device: Amplitude 80%, 30s intervals, Pulse 1.0 second on, 1.0 second off. However, using a more powerful sonicator is recommended to accelerate C. elegans fragmentation.

• 36. It is advised to perform a few test reactions prior to processing the entire batch for both quantification methods to be able to adjust sample dilution should it be too concentrated.

4.3. Cuticle (extracellular matrix) isolation in C. elegans

• 37. C. elegans tend to stick on the plastic tip or on the plastic tubes. Hence, make sure to use a glass Pasteur pipette and pipette the C. elegans on the bottom of the plastic tube to avoid sticking on the side of the tube.

• 38. If the cuticles in the freezing protocol are ruptured too much, you can consider performing only the freezing and thawing cycles or only sonication.

• 39. One of the major problems is losing too many cuticles or C. elegans. Therefore, use always glass pipettes to transfer worms and cuticles.

• 40. Triton-X prevents the cuticles from sticking to the tubes during the washing steps.