Eosinophils staining

[Cyn]

Hi.I'm trying to stain murine eosinophils from cytospin preparations. I'm using Dff-Quik that is a modified version of Wright technique. I've got many problems to obtain a clear preparation of eosinophls, with their red cytoplasmic granules and their multilobulled nucleus because I don't know if I'm washing in excess and my eosinophils get unstained. Please if someone has experience I will aprecciate any help. Thanks

[Piersgb]

Hello! I am also working with murine eosinophils, or trying to. What is the source of your murine eosinophils? Blood? Bone marrow? Organ?

 

I've been using the Diff-Quik kit also (from Gamidor?) where you dip your slide into the fixing, red, blue and then wash solutions. It did initally take some time to get some good stains but what I've concluded is that murine eosinophils stain much less vividly than human cells. I've had some cytospins of purified human eosinophils taken from the blood and using the same dipping protocol these stained amazingly.

 

I do:

- 10x1second dips in the fixing solution

- 15x1second dips in the red solution

- 8x1second dips in the blue solution

- 2xslow dips in the wash solution.

 

Another thing to watch out for is the pH of your wash solution. You get better eosinophillic staining if the wash solution is at a pH of around 6.6-6.8 (more acidic).

 

I don't think you could really wash the dyes out of the cells. The washing should really only remove excess dyes that haven't entered the cells. I think it may just be a case of having to look really carefully for red staining granules (I find difficult to see) but the nuclei should stain just fine.