Two-Photon Laser-Mediated Ablation of Osteoblasts in Developing Zebrafish Larvae

Podsumowanie

This protocol describes a two-photon laser ablation approach carried out in zebrafish larvae, which serves as a model to study bone regeneration and the effects of the immune response to ablation.

Streszczenie

Zebrafish (Danio rerio) have an outstanding capacity to regenerate different organs and appendages. Bone regeneration in zebrafish has been studied using different methods such as fin amputation, scale plucking, skull trepanation, and microscopic approaches. Using a confocal laser scanning setup equipped with a two-photon laser, a laser ablation method was developed as a lesion paradigm to ablate bone-forming cells (osteoblasts) in the developing opercle of zebrafish larvae. The method described here allows the ablation of cells in a precise manner, as the area, shape, and depth can be finely adjusted. In addition, this method allows imaging of the area before and just after the ablation, so that short-term effects of the injury can be analyzed. In this experimental setup, the immune response after ablation of osteoblasts in the injured area was studied. An increase in the recruitment of macrophages was observed after ablation, indicating the relevance of their presence during bone regeneration.

Wprowadzenie

Zebrafish regenerate diverse organs such as the retina, brain, heart, and pancreas¹. In addition, zebrafish regenerate skeletal elements, which is why they have been used to study the regeneration of fins, scales, and calvariae (skull caps)². Different experimental paradigms have been used to study tissue and bone regeneration, such as fin resection (amputation), fin fracture, skull trepanation^3,4, cryoinjury^5,6, or genetic ablation^7,8,....

Protokół

The study protocol received approval from the Landesdirektion Sachsen, permit numbers 25-5131/564/2, DD25-5131/450/4, 25-5131/496/56, DD25.1-5131/354/87. The zebrafish strains used were maintained according to national law and under standardized conditions as previously described^27,28. The details of all the reagents and the equipment used in the study are listed in the Table of Materials.

1. Preparation of materials and solutions

1. Maintain the embryos in 1x E3 media. Prepare the 1x E3 media by diluting 170 mL of a 60x E3 stock in 10 L of....

Dyskusje

Laser ablation has been applied in various disciplines of biological research. In particular, it has been useful as a method to study tissue regeneration^10,11,12. For example, the recruitment and phenotype changes of innate immune cells were recently analyzed over time using UV laser or two-photon laser ablation assays, along with the recovery of osteoblasts at the laser ablation site¹⁰. Live imaging of i.......

Materiały

Name	Company	Catalog Number	Comments
Calcium chloride dihydrate, CaCl2·H2O	Carl Roth	5239.1
Cell Culture Dish, PS, 100/20 mm	Greiner Bio-one	664160
Dumont #55 Foceps	FST	11295-51	Tip shape straight, 11 cm, 0.05 x 0.02 mm
Falcon 6-well plate	Corning	353502
Glass-bottom microwell dish	MatTek	P35G-1.5-14-C	35 mm Dish, No. 1.5 Coverslip, 14 mm Glass Diameter, Uncoated
Insight X3 multiphoton laser	Spectra-Physics
Leica Application Suite	LAS X, Leica Microsystems
Low melting agarose	Biozyme	840101	Biozym Plaque Agarose
Magnesium sulfate heptahydrate, MgSO4·7H2O	Sigma-Aldrich	M5921
Mating cages			many varieties, e.g. Tecniplast
Methyleneblue	Carl Roth	A514.1
MS-222	SIGMA Aldrich	A5040
Potassium Chloride, KCl	PanReac AppliChem	131494
Sodium chloride, NaCl	Carl Roth	3957.1
SP8 FALCON	Leica Microsystems		Equipped with a Insight X3 multiphoton laser and Leica Application Suite software
Stainless Steel Dissect Needle	Bochem	12010	140 mm
Stereo Microscope System SZX16	Olympus		Equipped with a LED illumination base SZX2-ILLTQ
Thermostatic Cabinets TS - WTW	xylem	TS 608/2-i	For incubation (embryo)
Transfer pipette, 3.5 mL	SARSTEDT	861171	155 x 15 mm, LD-PE, transparent
Zeiss SteREO Discovery.V12 version 4.7.1.0.	Zeiss		Equipped with Axiocam MRm camera and AxioVision sofware