In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

Summary

This protocol offers a method to study cellular dynamics using a simple in vitro culture technique. It provides an opportunity for zebrafish researchers and educators to study cellular processes, such as those related to bone homeostasis and basic cell biology, by visualizing fluorescent nuclei and apoptotic cells within the scales.

Abstract

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

Introduction

In recent years, zebrafish (Danio rerio) have emerged as a valuable model organism in various scientific fields, including genetics, developmental biology, and toxicology^1,2,3. Zebrafish are small tropical freshwater fish native to the rivers of Southeast Asia⁴. They have become a popular model organism in biology due to their easy maintenance, small size, rapid reproductive cycle, and translucent embryos¹. This allows for easy observation of their internal development, making them ideal for studying various biological processes in developmental biology. Zebrafish share genetic similarities with humans; approximately 70% of human genes have at least one zebrafish counterpart, making them an excellent model for studying genetic disorders and diseases in humans^5,6. By manipulating specific genes in zebrafish, researchers can gain valuable insights into the function and behavior of genes, which can then be applied to human health research⁶, gene editing, and the creation of transgenic lines^7,8.

Zebrafish scales regenerate after removal^9,10 and can be cultured for one to three days with a basic (non-CO₂) incubator while they are used for experiments¹¹. The use of zebrafish scales is advantageous in basic laboratories as they can be removed from anesthetized fish without the need for euthanasia. Zebrafish scales are a component of the dermal skeleton, consisting primarily of two layers: the internal layer, which is thick and made of partially mineralized tissue formed by layers of collagen fibrils, and the external layer, which is highly mineralized and contains a network of interwoven collagen fibrils^12,13 (Figure 1A-C). These morphological and cellular features of the scale are easily observed under a standard compound microscope. These characteristics make zebrafish scales a good model for studying cellular and molecular. For example, zebrafish scales have been used to study bone homeostasis, including bone cell activity^11,12. They have also been used to understand tissue regeneration^9,14,15, gene pathways and signaling^14,16, metabolism¹⁷, microbiota studies¹⁸, etc. Zebrafish, similar to other teleosts, are also highly sensitive to environmental factors such as pollutants and toxins^19,20,21,22. As such, fish scales are used to study exposures to toxins in fish populations. These characteristics make scales an excellent model for teaching students about the impacts of environmental factors on living organisms. Additionally, scales from transgenic zebrafish lines, such as Tg(osterix:mCherry) and Tg(runx2a:GFP), could add another level of complexity to student experiments.

In summary, by using zebrafish scales, researchers can design and conduct experiments to study various aspects of biology, from cellular mechanisms to environmental changes. Zebrafish are also a valuable resource in higher education since they can provide hands-on experiences in experimental design, data collection, and analysis. Zebrafish can also be utilized in advanced courses and research-focused programs to explore specific areas of interest. This article describes a protocol for using zebrafish scales in research and classroom learning environments.

Protocol

All protocols described here follow the Canadian Council on Animal Care guidelines and were approved annually by the Saint Mary's University/Mount Saint Vincent University Animal Care committee under protocol numbers 20-14 and 21-12. Before carrying out this protocol, users must ensure that federal and provincial guidelines on using animals in research or teaching are followed²³. The details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Preparation of reagents for scale culture

1. Prepare DMEM medium with HEPES (10 g of DMEM powder, 5.95 g of HEPES, in 1 L of distilled water or dH₂O, pH: 6.91), aliquot, and store at 4 °C.
2. Prepare the culture medium - working solution (88% DMEM medium with HEPES, 10% fetal bovine serum, and 2% penicillin-streptomycin [5000 units penicillin and 5 mg streptomycin per mL] adjusted to the volume needed on the day the experiment is performed.
   NOTE: The working solution must be prepared fresh daily and cannot be stored. The culture medium must be kept cold (at 4 °C or on ice) until used.
3. Aliquot the culture medium working solution into the containers in which the experiment will be carried out. In this case, 0.2 mL tubes or a 96-well plate were used. Maintain these aliquots on ice until required.
   NOTE: This solution should be prepared fresh and cannot be stored for more than 1 day. Refer to Table 1 for all reagents.

2. Removing scales from live fish

1. Prepare a container or tank for the zebrafish with a water volume of approximately 2 L for 4-5 adult fish.
   NOTE: The typical parameters of the water that are appropriate for zebrafish are pH 7.5, temperature 28.5 °C, conductivity 640-781 µS, and a 12-12-h dark-light cycle. Ensure that temperature is constantly maintained in the tank.
2. Use a fish net to capture 4-5 adult zebrafish and place them in the zebrafish holding tank prepared above.
3. Prepare a container with at least 60 mL of water where the fish will be anesthetized.
   NOTE: The fish must be able to move in the container and be deep enough to prevent them from jumping out. This container should contain buffered 0.01% tricaine methane-sulfonate (MS222), which should be made up of zebrafish water (Dilute 0.1% MS222, which is prepared with 0.3 g of MS222, 615 µL of 1 M NaOH in 300 mL of zebrafish water system, ten times).
   CAUTION: Individuals conducting these experiments must have the institutional animal ethics board's approval to catch, anesthetize, and handle zebrafish. Similarly, the anesthetic used is a low concentration of tricaine methane-sulfonate (MS222), which is a chemical commonly used in fish biology but one that requires proper handling, such as appropriate personal protective equipment, including disposable nitrile gloves, safety glasses/splash goggles and lab coat (Refer to the safety data sheet of MS222 for more information).
4. Prepare a recovery container with the same specifications as used previously in step 1 (at least 2 L of water) where the fish will be placed after scale removal. This container should have enough space and the correct water parameters, as noted above.
5. Using a net, carefully transfer a single adult zebrafish to the container with 0.01% MS222. Wait until the fish is completely immobile and has minimal opercular movement (this usually takes less than 10 min).
   NOTE: This entire step is carried out one fish at a time. Do not anesthetize the next fish until the scales have been successfully removed from the fish that is being handled and until that fish has been transferred to the recovery container.
6. Individually place the adult zebrafish on an inverted Petri dish lined with a wet paper towel.
7. Under a dissecting stereomicroscope and using fine forceps, remove a maximum of 10-12 scales from an individual fish. The scales are removed from the lateral region of the fish, where the scales are bigger and have a more constant size compared with the other parts of the body (Figure 2). Both sides of the fish can be used.
   1. Remove the scales one at a time by gently tugging in the natural direction of the scale (i.e., in the direction from anterior to posterior). This should be done as quickly as possible.
8. Place each individual scale in a separate 0.2 mL tube containing the scale culture medium-working solution (prepared in step 1.1) to prevent the scales from degradation. A 96-well plate can be used as an alternative.
   NOTE: Isolation of individual scales in separate tubes or wells prevents the scales from sticking together and allows the tracking of individual scales that may be needed for multiple experiments.
9. If the specific experiment requires a scale treatment, apply it in this step. For examples of some protocols, refer to steps 4 and 5.
10. Put the tubes with the scales inside an incubator at 28 °C. The culture medium should be changed every 24 h (with the culture medium-working solution). A non-CO₂ incubator was used for the experiments outlined here.
11. After scale removal, carefully return the zebrafish to the recovery tank and monitor until movement resumes. A pipette or aeration stone is used to provide air in the tank to speed up recovery.
12. Once movement is restored, return the zebrafish to a regular rearing tank. Remember to keep the fish that were used separately from any other fish so that they can recover separately and to keep track of which fish are actively regenerating their scales.
13. Wait ~2 weeks before repeating scale removal from these same fish. This allows time for them to regenerate their scales naturally.

3. Fixation of scales

1. Wash the scales three times with 200 µL of 1x of phosphate-buffered saline (PBS) prepared from 10x PBS (for 1 L, add 80 g of NaCl, 2 g of KCl, and 11.2 g of Na₂HPO₄, add dH₂O until 1 L).
2. Remove the 1x PBS and add 200 µL of 4% paraformaldehyde (PFA) overnight at 4 °C. For 100 ml of PFA, add 4 g of paraformaldehyde and 0.1 g of NaOH, then add 1x PBS until 100 mL. Mix on a hot plate set to low heat and stir until the solution is clear, then allow the solution to cool down and stabilize the pH to 7.4. Store at -20 °C. For details, please see Supplementary File 1 (Table 1).
3. After fixation, wash the scales three times with 200 µL of 1x PBS. Store the scales at 4 °C.
   CAUTION: PFA is harmful. Refer to the safety data sheet for more information. All these processes require proper handling, such as appropriate personal protective equipment, including disposable nitrile gloves, safety glasses/splash goggles, and a lab coat.

4. Bone homeostasis

NOTE: Two bone homeostasis procedures are described below. Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphate (AP) staining are typically used to identify active osteoclasts and osteoblasts, respectively. Osteoclasts resorb bone matrix while osteoblasts deposit bone matrix. AP is the main enzyme released by osteoblasts during bone formation, while TRAP is secreted by osteoclasts into the acidic space between the osteoclast and bone matrix, assisting in bone breakdown and resorption^24,25,26. This protocol is also available on The Zebrafish Information Network²⁷ and was previously published²⁸. Alkaline phosphatase is an enzyme produced by multiple cell types as part of the metabolic process. This process is not wholly specific to osteoblasts, but in bone tissue, it is used as a marker for osteoblast function.

1. Tartrate-resistant acid phosphatase (TRAP) staining
   1. Place each fixed scale into a 0.2 mL tube or a 96-well plate with 200 µL of dH₂O. Wash the scales by adding and removing around 150 µL of dH₂O for 15 min three times. For details, please see Supplementary File 1 (Table 2).
   2. Remove the scales from the 0.2 mL tube or well and place them in a new tube that contains 200 µL of 50 mM tartrate buffer. Incubate for 1 h at room temperature.
      1. For 100 mM of tartrate buffer, prepare a glass container, add 2.3 g of tartaric acid, and mix it with 0.2 M acetate buffer (pH 5.5) until 100 mL of solution. Stir well and store at 4 °C. To prepare 50 mM of tartrate buffer, add equal volumes of dH₂O and 100 mM tartrate buffer. For details, please see Supplementary File 1 (Table 3 and Table 4).
   3. Remove the tartrate buffer from the tube and add 200 µL of TRAP substrate solution. Incubate the samples for 1 h at room temperature. Ensure samples remain in the dark.
      1. Prepare a dark glass container and add 30 mL of 100 mM tartrate buffer, 1 mL of hexazotized PRS (Pararosaniline hydrochloride solution), and 2 mL of enzyme substrate solution. Stir well.
         NOTE: This solution cannot be stored; keep it in the dark at room temperature while using. For the solutions needed to prepare the TRAP substrate solution, please see Supplementary File 1 (Tables 5-9).
   4. Remove the TRAP substrate solution and wash the scales by adding and removing around 150 µL of dH₂O for 15 min, three times.
   5. Remove the scales from the tube and place them into a new tube or well with 200 µL of 80% glycerol (for 100 mL, mix 80 mL of glycerol with 20 mL of dH₂O) wrapped in aluminum foil to avoid fading and store them at 4 °C.
   6. To visualize the scales under the microscope, use a depression or concave slide.
2. Alkaline phosphate (AP) staining
   1. Place each fixed scale in a 0.2 mL tube or 96-well plate with 200 µL of dH₂O and wash them by adding and removing around 150 µL of dH₂O for 15 min, three times. For details, please see Supplementary File 1 (Table 2).
   2. Remove the scales from the 0.2 mL tubes or wells and place them into a new tube that contains 200 µL of tris-maleate buffer and incubate it for 1 h at room temperature.
      1. For this buffer, prepare a glass container and add 0.605 g of tris buffer and 0.55 g of maleic acid. Add dH₂O until 25 mL and stir well. Stabilize to pH 8.3. Store at 4 °C. For details, please see Supplementary File 1 (Table 10).
   3. Remove the tris-maleate buffer from the tube and add 200 µL of AP substrate solution. Incubate it for 1 h at room temperature. Ensure samples remain in the dark.
      1. For this solution, prepare a dark glass container, add 8 mg of diazonium salt, and mix it with 0.1 mL of napthol-AS-TR-phosphate in N,N-dimethylformamide, and 10 mL of tris-maleate buffer (pH 8.3), stir well. Prepare this solution fresh. For details, please see Supplementary File 1 (Table 11).
   4. Remove the AP substrate solution and wash the scales by adding and removing 200 µL of dH₂O for 15 min, three times.
   5. Remove the scales from the tube and place them into a new tube or well with 200 µL of 80% glycerol wrapped in aluminum foil to avoid fading. Store at 4 °C.
   6. To visualize the scales using a microscope, place the scales on a concave or depression slide.
      CAUTION: Most of the reagents used in the TRAP and AP staining can be harmful. Refer to the safety data sheet for each chemical. All these procedures require proper handling, such as appropriate personal protective equipment, including disposable nitrile gloves, safety glasses/splash goggles, and a lab coat.

5. Visualizing cell dynamics in zebrafish scales

NOTE: A range of staining methods can be used to visualize differences between scales and to label cells within the scale (Figure 3B). For example, methods to stain nuclei and lysosomes within the cells of the scale are outlined below.

1. DAPI staining for cell nuclei visualization
   NOTE: DAPI is used to mark the nuclei of all cells by labeling the DNA. DAPI is a fluorescent marker that binds strongly to regions enriched for adenine and thymine in DNA sequences.
   1. Before using DAPI, ensure that scales are fixed in 4% paraformaldehyde (step 3). Wash the scales with 200 µL 1x PBS for 5 min after fixing.
   2. Prepare a DAPI working solution in a 1:10 dilution as follows. Mix 1 µL of DAPI with 9 µL of 1x PBS to make a total volume of 10 µL.
   3. After preparing the DAPI solution, use forceps or a plastic pipette to put the scale on a concave slide, remove excess PBS, and add around 10 µL of the prepared DAPI solution.
      NOTE: DAPI will stain the scale immediately, so it can be visualized using a fluorescence microscope with an excitation wavelength of 352-402 nm and an emission wavelength of 417-477 nm. DAPI will stain all cell nuclei a bright blue color. In order to see the entire scale in the field of view, the 4x objective was used. Single stained nuclei are visible using the 20x objective.
   4. Count the number of DAPI-stained nuclei in order to determine the number of cells in the scale.
2. Apoptotic staining for cell death visualization in live samples
   NOTE: Fluorescent stains can be used to label apoptotic cells. For example, the fluorescence marker protocol used here is for acidic environments and can be used for labeling and tracing acidic organelles like lysosomes. An increase in the number of these organelles is correlated with increased cell death. This stain should be applied to unfixed scales that are either freshly pulled or have been cultured for up to 2 days.
   1. Prepare a solution of the fluorescent stain for apoptosis tracking according to the manufacturer's instructions by using the culture medium as the dilutant.
      NOTE: The concentration of the fluorescent stain can change drastically depending on the apoptosis method used. Refer to the commercial protocol for the stain of choice for specific details on recommended concentrations.
   2. Carefully remove the culture medium from each 0.2 mL tube or well from a 96-well plate if the scales were previously cultured.
   3. Add enough fluorescent staining solution to cover the whole scale (around 50 µL).
   4. Cover all the tubes with aluminum foil to avoid fading and leave for 30 min.
   5. Remove the fluorescent staining solution from the tubes or wells.
   6. Wash samples by adding 200 µL of 1x PBS to the tubes or wells for 5 min.
   7. Remove the PBS and add 200 µL of 4% PFA to the tubes or wells to fix the samples for 1 h at room temperature.
   8. Wash samples by adding 200 µL 1x PBS to the tubes or wells for 5 min.
   9. Transfer the scales into a new 0.2 mL tube or well, and add 200 µL of fresh 1x PBS. Cover these tubes/wells in aluminum foil to avoid fading. Store at 4 °C.
   10. To visualize the scales with a fluorescence microscope, use a concave or depression slide, and the correct filter. The fluorescent signal used here has an excitation and emission maximum of 577-590 nm. The fluorescent stain shows apoptotic cells containing lysosomes in red.
   11. Count the number of stained cells in order to determine the number of cells undergoing apoptosis.
       NOTE: Aluminum foil is used during staining and storage to avoid fading. Samples can be stored in the dark for a couple of hours or even days, depending on which fluorescent stain is used. To store fluorescent samples for longer periods, use a fixation and mounting protocol that is compatible with the fluorescent stain. All fluorescent staining was visualized using a fluorescence microscope with at least 20x magnification. The filter used was as follows: for DAPI 417-477 nm; for apoptosis tracking 577-590 nm.

Results

Recently, this scale culturing protocol was used to study bone homeostasis¹¹. Scales can survive in the culture medium for at least 2 days. The numbers of apoptotic cells were analyzed on each day of culture, up to a maximum of three days, by counting the labeled cells. These results showed that apoptotic cells increased significantly on the third day of culturing, indicating that culture time should be limited to two days using the method described here.

Comparing scal...

Discussion

Zebrafish scales are an easily accessible in vitro model to study a variety of different biological processes that can be maintained for up to 2 days using a culturing method and an incubator to simulate their natural environment¹¹. Scales have a regular and proportional distribution of cells present, which enables researchers to view, count, and label cells and to conduct simple cell biology experiments using basic laboratory equipment. Furthermore, the entire scale is easily visible wit...

Materials

Name	Company	Catalog Number	Comments
0.2 mL tubes	n/a	n/a
37% HCl	EMD	HX0603-4	For pH stabilization and prepration of PRS
Concave slide	n/a	n/a
DAPI	Vectashield	H-1200	For cell nuclei visualization
Diazonium salt (Fast Blue B)	Sigma	D9805	For AP substrate solution
Dulbecco's Modified Eagle Medium (DMEM) powder	Sigma	D5523	For scale culture media
Ethyl 3-aminobenzoate methanesulfonate (MS222)	Sigma	E10521	For preparation of 0.1% MS222
Fetal bovine serum	Sigma	F4135	For scale culture media
Fluorescence microscope	n/a	n/a
Glacial acetic acid	Fisher	A38 212	For acetate buffer
Glycerol	VWR	BDH1172-4LP	For 80% glycerol - storage scales
HEPES	ThermoFisher	15630106	For scale culture media
Hot plate	n/a	n/a
KCl	Sigma	P217-10	For preparation of PBS
Lysotracker	ThermoFisher	L7528	For lysosomes  visualization
Maleic acid	Sigma	M0375	For Tris-Maleate buffer
Micropipette	n/a	n/a
N,N-dimethylformamide	Sigma	319937	For AP substrate solution
N,N-dimethylformamide	Sigma	319937	For Enzyme Substrate Solution
Na2HPO4	EMD	SX0720-1	For preparation of PBS
NaCl	Sigma	S9888	For preparation of PBS
NaNO2	Sigma	S-2252	For 4% NaNO2
NaOH	Sigma	S5881	For preparation of 4% PFA
NaOH	Sigma	S5881	For scale fixation and pH stabilization
Napthol-AS-TR-phosphate	Sigma	N6125	For AP substrate solution
Napthol-AS-TR-phosphate	Sigma	N6125	For Enzyme Substrate Solution
Pararosaniline hydrochloride	Sigma	P3750	For PRS
Penicillin-streptomycin 5000 units penicillin and 5mg streptomyocin/ml	ThermoFisher	15140122	For scale culture media
Personal protective equipment (PPE): disposable nitrile gloves, safety glasses/splash goggles and lab coat.	n/a	n/a
PFA	Sigma	P6148	For scale fixation
Sodium acetate	Sigma	S2889	For acetate buffer
Standard compound microscope	n/a	n/a
Stir plate	n/a	n/a
Tartaric acid	Sigma	T6521	For Tartrate buffer
Tris base	Roche	03 118 142 001	For Tris-Maleate buffer