Apr 02, 2020

Public workspaceStaining of fish Red Blood Cells

DOI
dx.doi.org/10.17504/protocols.io.bd9yi97w
  • Robson Andrade Rodrigues1,
  • Mayara Schueroff Siqueira1,
  • Brenda Oliveira Martins1,
  • Carlos Eurico Fernandes1,
  • Lilian Franco-Belussi1,
  • Diogo B. Provete1
  • 1Universidade Federal de Mato Grosso do Sul
Diogo B. Provete
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DOI: dx.doi.org/10.17504/protocols.io.bd9yi97w
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
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,
Abstract
Materials
MATERIALS
ReagentMethanolP212121Catalog #PA-33900HPLCCS4L
Reagent100ml Giemsa Stain Stock SolutionG-BiosciencesCatalog #786-1065
Reagent20 mg EugenolbiorbytCatalog #orb104769
ReagentWright's stain (Eosin methyl blue)Bio Basic Inc.Catalog #WB0989.SIZE.25g
Reagent 1 ml syringes (or U-100 Insulin Syringe)BD BiosciencesCatalog #329461
ReagentEDTAThermo FisherCatalog #17892
ReagentShandon™ Wright-Giemsa Stain Kit, Wright-Giemsa solutionThermo FisherCatalog #9990710
STEP MATERIALS
ReagentEthylenediaminetetraacetic acidSigma – AldrichCatalog #E9884
Reagent20 mg EugenolbiorbytCatalog #orb104769
You can make your own MGGW stain solution or buy ready-to-use kits.
Protocol materials
Reagent20 mg EugenolbiorbytCatalog #orb104769
In Before starting, Materials, Materials
Reagent 1 ml syringes (or U-100 Insulin Syringe)Becton Dickinson (BD)Catalog #329461
Materials
ReagentMethanolP212121Catalog #PA-33900HPLCCS4L
Materials
Reagent100ml Giemsa Stain Stock SolutionG-BiosciencesCatalog #786-1065
Materials
ReagentWright's stain (Eosin methyl blue)Bio Basic Inc.Catalog #WB0989.SIZE.25g
Materials
ReagentEthylenediaminetetraacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #E9884
Materials, Step 1
ReagentEDTAThermo FisherCatalog #17892
Materials
ReagentShandon™ Wright-Giemsa Stain Kit, Wright-Giemsa solutionThermo FisherCatalog #9990710
Materials
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 (Amount50 mg /L)

Reagent20 mg EugenolbiorbytCatalog #orb104769
.

Blood extraction
Blood extraction
Prepare a Amount1 mL syringe with Concentration3 % volume EDTA.

Note
Syringe spec is 24 G x 3/4” (20 mm x 0.55 mm)

ReagentEthylenediaminetetraacetic acidSigma AldrichCatalog #E9884

Dilute Amount3 g of EDTA powder in Amount100 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
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 dryDuration00:20:00

20m
Staining
Staining
Take about Amount300 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 Amount1 g of eosin methyl blue of May-Grünwald, Amount1 g of eosin methyl blue of Giemsa, and Amount1 g eosin methyl blue of Wright, diluted in Amount1 L of methanol. The stock solution should be kept at Temperature25 °C for at least 1 week before use. Then, you should filter it and it's ready to be used.
ReagentMethanolP212121Catalog #PA-33900HPLCCS4L

Reagent500ml Giemsa Stain Stock SolutionG-BiosciencesCatalog #786-1066

ReagentWright's stain (Eosin methyl blue)Bio Basic Inc.Catalog #WB0989.SIZE.25g






Wait for Duration00:03:00

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 Duration00:10:00

10m
Wash slides in tap waterDuration00:00:15

10s
Wait slides dryDuration00:20:00

20m
Analyse cells in image analysis software, such as ImageJ or Motic Image Plus
Software
FIJI (Image J)
NAME
NIH
DEVELOPER

Software
Motic Images Plus
NAME
Windows and macOS
OS
Motic
DEVELOPER