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LUNA Staining Protocol to detect eosinophils in tissue

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#1 Empath



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Posted 23 March 2015 - 02:42 AM

Hi  everyone.

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.




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