Protocol Citation: Robson Andrade Rodrigues, Mayara Schueroff Siqueira, Brenda Oliveira Martins, Carlos Eurico Fernandes, Lilian Franco-Belussi, Diogo B. Provete 2020. Staining of fish Red Blood Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bd9yi97w
MANUSCRIPT CITATION:
Martins, B. O.; Franco-Belussi, L.; Siqueira, M. S.; Fernandes, C. E. S.; Provete, D. B. (2020): The evolution of red blood cell shape in a continental radiation of fishes.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
This is a technique to extract blood and prepare blood films of fish to make stereological analysis of blood cells. This is a modification of Rosenfeld (1947) technique by Tavares-Dias and Moraes (2006) using common staining techniques and is called May-Grünwald-Giemsa-Wright.
Dilute 3 g of EDTA powder in 100 mL of distilled water
2
Extract blood from the vena caudalis or by cardiac puncture (for large fish specimens), or by decaptation (for small specimens)
Note
If you have small fish specimens, you don't need the syringe. You just have to use 1 µL pippete to get one drop of blood from the head of fish.
Cardiac puncture of small fish specimens
Preparing blood film
3
Place one blood drop on a microscope slide
Placing blood drop on the slide
4
Take an extra microscope slide, touch the blood drop at 45º and then slide it until the drop is fully spread
Begining of the procedure showing how to position the extra slide
20m
5
Let the slide dry00:20:00
Staining
6
Take about 300 µL of the MGGW stain solution and drop it on the slide so all the slide is covered.
Note
The stock solution of the May-Grünwald-Giemsa-Wright (MGGW) stain is made with 1 g of eosin methyl blue of May-Grünwald, 1 g of eosin methyl blue of Giemsa, and 1 g eosin methyl blue of Wright, diluted in 1 L of methanol. The stock solution should be kept at 25 °C for at least 1 week before use. Then, you should filter it and it's ready to be used.
