[Histonet] AFB staining on mouse tissue

[Connie McManus]

Your're right. if you try to use a staining procedure on tissue sections
that is usually done on a smear, it won't work.  Tissue staining usually
requires more time, and sometimes entirely different reagents.  This is
what I use in my lab.  Reference is AFIP, 1992 edition
 
SOLUTIONS:
Carbol Fuchsin
      Phenol (melted) ............... 2.5 mL
      100% Ethanol .................. 5 mL
      Basic Fuchsin (CI 42500) ...... 0.5 g
      DI water ...................... 50 mL
      Mix in the order given.  Filter before use.
 
Methylene Blue (Stock solution)
      Methylene Blue (CI 52015) ..... 1.4 g
      DI water ...................... 100 mL
 
Methylene Blue (working solution)
      Stock Methylene Blue ........... 5 mL
      Tap water ...................... 45 mL
 
Acid Alcohol
      70% Ethanol ................... 99 mL
      HCl, conc ..................... 1 mL
 
PROCEDURE:
Preheat Carbol Fuchsin in a 60 C oven then swirl well BEFORE filtering
(this is my own addition to this procedure). 
      1.   Cut sections at 4-5 um
      2.    Deparaffinize as usual 
      3.    Rinse well in DI water
      4.    Carbol Fuchsin, 30 minutes at Room Temp
      5.    Rinse in running tap water to remove excess dye.
      6.    Dip in acid alcohol until sections turn a pale pink
      7.    Running tap water, 5 minutes
      8.    Working methylene blue,  1 or 2 dips.  DO NOT OVER DO THIS
STEP!!!
      9.    Dehydrate through graded alcohols to xylene or xyl
substitute
      10.   Mount in the appropriate resin
 
Hope this is helpful
 
Connie McManus
Utah Veterinary Diagnostic Laboratory
Utah State University
Logan, UT