Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Principle

A differential visualisation of tissue elements is achieved with two similar acid dyes. A dye with a small molecule size (Biebrich scarlet/acid fuchsin) is used first which stains all tissue elements in the section. This red stain is selectively removed from unwanted areas by differentiating with phosphomolybdic acid, which also acts as a mordant for the next step of the procedure. A dye with a large molecule (light green) is then progressively applied to the section. A celestin blue/haematoxylin is used to stain the nuclei as it is more resistant than alum haematoxylins to removal by the acid solutions used in the method. This technique differentiates oedema from fibrosis and visualises immunological deposits. Post mordanting in bouins before staining with this method, gives enhanced acidophilic staining.

Technical Points

1.      (step 8) - The mordanting by dodecamolybdophosphoric acid is easily removed by washing

Method

1.      Sections to distilled water

2.      Stain in Celestin blue 5 mins

3.      Rinse in distilled water

4.      Stain nuclei with alum haematoxylin 5 mins

5.      Wash in running tap water 5 mins

6.      Rinse in distilled water

7.      Stain with Biebrich scarlet-acid fuchsin stain 10 mins

8.      Rinse in distilled water

9.      Treat with freshly prepared dodecamolybdophosphoric acid 10 mins

10.    Drain, do not rinse

11.    Stain with light green 10 mins

12.    Rinse excess stain from slide with alcohol

13.    Dehydrate, clear and mount.

Results

         fibrinoid.................................red

         immune deposits.......................red

         basement membrane..................green

         collagen.................................green

         nuclei....................................blue

Reagent Formulae

1.  Celestin blue

2.  Biebrich scarlet

                        Biebrich scarlet (C.I. 26905)         0.9 g

                        Acid fuchsin (C.I. 42685)              0.1 g

                        Glacial acetic acid (Analar)           1.0 ml

                        Distilled water                             100 ml

Mix the powders together then dissolve in distilled water. Add acetic acid.

2.  5% aq dodecamolybdophosphoric acid (Phosphomolybdic acid)

                   Prepare weekly

                  Phosphomolybdic acid                5 g

                  Distilled water                          100 ml

4.  1% Light Green in 4% Acetic Acid stain
                              light green SF (CI 42095)            1.0 g
                              distilled water                           96.0 ml

                              Glacial Acetic Acid                     4.0 ml
      Dissolve the dye in the water, add Acetic Acid.  Mix well. The stain keeps well.

References

Masson PJ, (1929), J.Technol.Methods