Zebrafish Erythrocyte Staining Protocol

Zebrafish embryos at 60 hpf were anesthetized by tricaine, then were immersed in mixture solution (40% FBS [HyClone] and a final concentration of 5 mM EDTA solution in 1X PBS). Red blood cells were collected from the heart using a microinjection needle. The cell solution was centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Then the enriched cells were smeared evenly on slide and air-dried rapidly. After fixing in the methanol for 5 min, the slide was soaked in rapid Wright–Giemsa staining solution (BBI Life Sciences) for 10 min. Finally, slides were rinsed with deionized water. Images were captured with 100× lens after air dry. Assessment of erythrocyte status was based on previously published article^{34 (link)}.

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Chen C., Lu M., Lin S, & Qin W. (2020). The nuclear gene rpl18 regulates erythroid maturation via JAK2-STAT3 signaling in zebrafish model of Diamond–Blackfan anemia. Cell Death & Disease, 11(2), 135.

Publication 2020

rapid Wright–Giemsa staining solution

Cells Edta Embryos Erythrocyte Heart Lens Methanol Microinjection Needle Tricaine Zebrafish

Other organizations : Peking University, University of California, Los Angeles

Top 5 similar protocols

Variable analysis

independent variables

Immersion of zebrafish embryos in a mixture solution (40% FBS [HyClone] and a final concentration of 5 mM EDTA solution in 1X PBS)

dependent variables

Erythrocyte status

control variables

Anesthetization of zebrafish embryos by tricaine
Centrifugation of the cell solution at 1000 rpm for 5 min
Fixation of the smeared cells in methanol for 5 min
Staining of the slides with rapid Wright–Giemsa staining solution for 10 min
Rinsing of the slides with deionized water

controls

Assessment of erythrocyte status based on a previously published article (link provided)

Annotations

Based on most similar protocols

The Input protocol specifies the use of 40% FBS (HyClone) and 5 mM EDTA solution in 1X PBS as the mixture solution to anesthetize and collect red blood cells from the heart of 60 hpf zebrafish embryos (Input protocol).

Protocol 1 describes an alternative method to collect peripheral blood cells from euthanized zebrafish by tail amputation in adults and heart puncture in embryos or larvae, followed by fixing the blood smears in 100% methanol for 15 seconds and staining using a Hema3 kit (an analog to Wright–Giemsa method) to visualize erythrocyte morphology. The protocol also includes a hemoglobin staining step using an o-dianisidine staining buffer (Protocol 1).

Protocol 2 outlines a method to prepare zebrafish erythroid cells from the heart or vessels at 52 hpf, attach them to slide glass, and perform Wright–Giemsa staining to label the erythroid cells. The protocol also describes a whole mount o-dianisidine staining method to detect hemoglobin in dechorionated zebrafish embryos at 48 hpf (Protocol 2).

Protocol 3 describes the use of a light sheet Z.1 microscope (Zeiss) to image anaesthetized zebrafish embedded in 1% low-melting point agarose, with the chamber containing E3 and tricaine (164 mg/L) to ensure the embryos and larvae remain anaesthetized throughout imaging. This protocol is focused on detecting blood flow and tracking endothelial cell nuclei, as well as imaging individual erythrocytes in gata1a morphants (Protocol 3).

Protocol 4 outlines a method to inject recombinant human erythropoietin (rhEPO) into the common cardinal vein of 48 hpf dechorionated zebrafish embryos, followed by incubation at 28°C for 2-4 hours and quantification of erythrocytes using o-dianisidine staining and hemoglobin quantification by a modified cyanomethemoglobin method. The protocol includes the use of phenol red solution as a negative control (Protocol 4).

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