This is the first time I'm posting.
I am trying to perform a Luna's protocol to detect eosinophils in formalin-fixed paraffin-embedded oral mucosa tissues.
I'm currently using the following protocol :
G-486 Luna’s method for Erythrocytes and Eosinophil Granules
· Deparaffinize and hydrate to distilled water
· Stain for 5 minutes in Working Hematoxylin –Biebrich Scarlet Solution
*To prepare Working Hematoxylin –Biebrich Scarlet Solution : First prepare Hematoxylin Working Solution by mixing:
1. Weigert’s Hematoxylin A 22,5 mL ( 1.2g Hematoxylin in 104,4 ml alcohol 100%)
2. Weigert’s Hematoxylin B 22,5 mL (4 ml Ferric chloride 30% ; 1 ml Hydrochloric acid 35% ; 95 ml MilliQ water)
(Mix solution well and add)
3. Biebrich Scarlet, 1% Aqueous 5 mL (Mix well)
· Differentiate in Acid Alcohol 1% until the desire nuclear detail is achieved (usually 8 dips). Remove Acid Alcohol by rinsing in water
· Dip in Lithium Carbonate 0.5% until section turns blue and erithrocytes are bright reds (usually 5 dips) Wash in running water for 2 minutes.
· Dehydrate in Alcohol and xilol
· Mount with DPX
Stain Results : Eosinophil granules and erythrocytes =Red
Background = Blue
I tried several time to get the expected results without any result : in the microscope i see everything blue. I tried to "play" with the concentration of fuchsin or with the incubation with acid alcohol or lithium carbonate but the result is always the same and i cannot see the red staining.
Anyone have any ideas? If anyone has a suggestion or an alternative protocol, please write me.
I'm looking forward to hearing from you soon.