Muskel und Fett - Aniline Blue-Orange G Stain for muscle fibers

Aniline Blue - Orange G Stain

This stain provides a good contrast between muscle fibers (orange), connective tissue (blue), and fat cells (white).

Procedure:

1. Cut tissue sample on a microtome 10 to 12 µm thick and place onto microscope slides (microscope slides should be pre-rinsed/cleaned in an ethanol solution). Leave slides at room temperature for at least 5 min.

2. Fix sections using Müller´s reagent for 1 min.

3. Rinse for 1 to 2 min in d₂H₂O.

4. Stain sections with Orange G for 3 min.

5. Rinse for 2 min in dH₂O (twice).

6. Stain sections with Aniline Blue for 1 to 3 seconds.

7. Rinse (with gentle shaking) immediately for 2 to 5 minutes in dH₂O (twice) (until no blue color is seen in the water)

8. Wash with: 70% Ethanol for 5 to 10 sec

96% Ethanol for 5 to 10 sec

absolute Ethanol for 1 to 2 min

9. Fix with Xylol for between 5 to 60 min (dispose of in proper receptacle).

10. Mount a cover slip using a non-aqueous cover slip medium (i.e. Histofluid).

Solutions and Reagents:

Müller´s reagent (Fresh Daily)

Potassium dichromate (K₂Cr₂O₇) 2.5 g

Sodium sulfate (Na₂SO₄-10H₂O) 1.0 g

dH₂O 100 mL

Phenol pinch (tip of a scupula, see notes)

Orange G solution (Store at room temperature, good for over 1 year)

Orange G 3.0 g

Acetic Acid, conc. 5.0 mL

dH₂O 100 mL

Bring the solution to a boil, cool to room temperature, and filter.

Aniline Blue solution (Store at room temperature, good for over 1 year)

Aniline Blue 0.5 g

Acetic Acid, conc. 5.0 mL

dH₂O 100 mL

Bring the solution to a boil, cool to room temperature, and filter.

Notes:

- The phenol added to the aniline blue/orange G stain is used as a preservative to stop bacterial growth. The recipe is over 100 years old and was used when water sources were not as clean as they are today. Imagine the end of a pen and use an amount of phenol equivalent to the tip of the pen. Its basically just a few grains.