TITLE: Staining of fish Red Blood Cells
AUTHORS: Robson Andrade Rodrigues, Mayara Schueroff Siqueira, Brenda Oliveira Martins, Carlos Eurico Fernandes, Lilian Franco-Belussi, Diogo Provete

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 </span><span style = "font-weight:bold;">May-Grünwald-Giemsa-Wright</span><span>. </span></div><div class = "text-block"><a href="https://bibliotecadigital.butantan.gov.br/edicao/memorias-do-instituto-butantan-tomo-xx-1947/335" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Rosenfeld, G. (1947) Corante pancrômico para hematologia e citologia clínica. Nova combinação dos componentes do May-Grünwald e do Giemsa num só corante de emprego rápido. Memórias do Instituto Butantan 20:329-334</span></a><span></span></div><div class = "text-block"><a href="http://www.seer.ufu.br/index.php/biosciencejournal/article/view/6443" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Tavares-Dias, M., & de Moraes, F. R. (2006). Caracteristicas hematologicas da Tilapia rendalli Boulenger, 1896 (Osteichthyes: Cichlidae) capturada em "pesque-pague" de Franca, Sao Paulo, Brasil. Bioscience Journal, 19(1): 107-114.</span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [Preparing blood film]
Let the slide dry
?. [Staining]
Wait for
?. [Staining]
Take about  of the MGGW stain solution and drop it on the slide so all the slide is covered.
300 µl
The stock solution of the May-Grünwald-Giemsa-Wright (MGGW) stain is made with   of eosin methyl blue of May-Grünwald,   of eosin methyl blue of Giemsa, and    eosin methyl blue of Wright, diluted in   of methanol. The stock solution should be kept at   for at least 1 week before use. Then, you should filter it and it's ready to be used.
?. [Staining]
Wait the stain dry for
?. [Staining]
Wash slides in tap water
?. [Blood extraction]
Prepare a  syringe with   EDTA.
1 ml
Syringe spec is 24 G x 3/4” (20 mm x 0.55 mm)
?. [Blood extraction]
Dilute  of EDTA powder in  of distilled water
3 g
100 ml
?. [Blood extraction]
Extract blood from the vena caudalis or by cardiac puncture (for large fish specimens), or by decaptation (for small specimens)
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.
?. [Preparing blood film]
Place one blood drop on a microscope slide
?. [Preparing blood film]
Take an extra microscope slide, touch the blood drop at 45º and then slide it until the drop is fully spread
?. [Staining]
Add drops of distiled water to the slide with stain to dilute the stain.
you can use a plastic straw or a pasteur pipette to homogeneize the solution on the slide.
A fine, shiny layer of stain precipitate will appear on the slide. This is because the stain does not dilute in methanol.
?. [Staining]
Wait slides dry
?. Analyse cells in image analysis software, such as ImageJ or Motic Image Plus