Apr 02, 2020
Staining of fish Red Blood Cells
- Robson Andrade Rodrigues1,
- Mayara Schueroff Siqueira1,
- Brenda Oliveira Martins1,
- Carlos Eurico Fernandes1,
- Lilian Franco-Belussi1,
- Diogo Provete1
- 1Universidade Federal de Mato Grosso do Sul
Protocol Citation: Robson Andrade Rodrigues, Mayara Schueroff Siqueira, Brenda Oliveira Martins, Carlos Eurico Fernandes, Lilian Franco-Belussi, Diogo 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
Protocol status: Working
We use this protocol and it's working
Created: March 26, 2020
Last Modified: April 02, 2020
Protocol Integer ID: 34840
Keywords: Erythrocytes, Blood, Blood cells, blood films of fish, fish red blood cell, caracteristicas hematologicas da tilapia rendalli boulenger, stereological analysis of blood cell, blood film, blood cell, fish, staining technique, blood, cell, cichlidae,
Abstract
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.
Materials
MATERIALS
MethanolP212121Catalog #PA-33900HPLCCS4L
100ml Giemsa Stain Stock SolutionG-BiosciencesCatalog #786-1065
20 mg EugenolbiorbytCatalog #orb104769
Wright's stain (Eosin methyl blue)Bio Basic Inc.Catalog #WB0989.SIZE.25g
1 ml syringes (or U-100 Insulin Syringe)Becton Dickinson (BD)Catalog #329461
EDTAThermo FisherCatalog #17892
Shandon™ Wright-Giemsa Stain Kit, Wright-Giemsa solutionThermo FisherCatalog #9990710
STEP MATERIALS
Ethylenediaminetetraacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #E9884
20 mg EugenolbiorbytCatalog #orb104769
You can make your own MGGW stain solution or buy ready-to-use kits.
Protocol materials
20 mg EugenolbiorbytCatalog #orb104769
1 ml syringes (or U-100 Insulin Syringe)Becton Dickinson (BD)Catalog #329461
MethanolP212121Catalog #PA-33900HPLCCS4L
100ml Giemsa Stain Stock SolutionG-BiosciencesCatalog #786-1065
Wright's stain (Eosin methyl blue)Bio Basic Inc.Catalog #WB0989.SIZE.25g
Ethylenediaminetetraacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #E9884
EDTAThermo FisherCatalog #17892
Shandon™ Wright-Giemsa Stain Kit, Wright-Giemsa solutionThermo FisherCatalog #9990710
Safety warnings
Methanol is volatile, so use a fume hood.
Before start
Before collecting blood samples, you need to anaesthetize fish using a eugenol solution (50 mg /L)
20 mg EugenolbiorbytCatalog #orb104769
.
Blood extraction
Prepare a 1 mL syringe with 3 % volume EDTA.
Note
Syringe spec is 24 G x 3/4” (20 mm x 0.55 mm)
Ethylenediaminetetraacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #E9884
Dilute 3 g of EDTA powder in 100 mL of distilled water
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
Place one blood drop on a microscope slide
Placing blood drop on the slide
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
Let the slide dry00:20:00
20m
Staining
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.
MethanolP212121Catalog #PA-33900HPLCCS4L
500ml Giemsa Stain Stock SolutionG-BiosciencesCatalog #786-1066
Wright's stain (Eosin methyl blue)Bio Basic Inc.Catalog #WB0989.SIZE.25g
Wait for 00:03:00
3m
Add drops of distiled water to the slide with stain to dilute the stain.
Note
you can use a plastic straw or a pasteur pipette to homogeneize the solution on the slide.
Expected result
A fine, shiny layer of stain precipitate will appear on the slide. This is because the stain does not dilute in methanol.
Wait the stain dry for 00:10:00
10m
Wash slides in tap water00:00:15
10s
Wait slides dry00:20:00
20m
Analyse cells in image analysis software, such as ImageJ or Motic Image Plus
Software
Motic Images Plus
NAME
Windows and macOS
OS
Motic
DEVELOPER