[Histonet] SOLVENT BLUE 38 ON PARAFFIN EMBEDDED MOUSE BRAIN

[Tim Wheelock]

Hi Nick:

I use Solvent Blue 38 (also known as Luxol Fast Blue) on formalin-fixed, 
paraffin embedded human brain, but have also seen it work well on mouse 
brain.
The protocol is as follows. This particular recipe combines the myelin 
stain with an H+E, but it can be combined with a Nissl stain instead or 
simply by itself.
Hope this helps.


Tim Wheelock
Harvard Brain Tissue Resource Center
McLean Hospital
617-855-3592


* LUXOL FAST BLUE**-HEMATOXYLIN-EOSIN (LHE) STAIN*

* *

1. De-paraffinize 5 micron formalin-fixed sections and take them down to 
95% ethanol.

2. Stain in Luxol Fast Blue solution for 2 hours at 60C.

3. Remove and allow to cool for 15 minutes.

4. Remove excess stain in 95% ethanol for 1 minute.

5. Wash in several changes of tap water.

6. Differentiate sections with 2 dips in the reducer solution, then 
*immediately* through 4

changes of tap water (10 dips each), then one more change of tap water.

7. Immerse sections in Gill 3 Hematoxylin for 10 minutes.

8. Rinse in several changes of tap water.

9. Differentiate sections in acid alcohol (7 dips).

10. Rinse in 4 changes of tap water (10 dips each) then one more change 
of tap water.

11. Blue sections in Scott’s Tap Water Substitute for 30 seconds.

12. Wash in 4 changes of tap water for 2 minutes each.

13. Immerse sections in 95% ethanol for 1 minute.

14. Immerse slides in alcoholic Eosin Y for 3 minute.

15. Differentiate in 95% ethanol (4 dips).

16 Dehydrate in first absolute ethanol for 20 dips.

17. Dehydrate in second absolute ethanol for 1 minute, and then clear, 
and mount.

*SOLUTIONS*: Luxol Fast Blue: Luxol Fast 
Blue...............................................0.1 gram.

10% glacial acetic acid .......................................1 mls.

95% ethanol........................................................100mls.

Reducer: 
Hydroquinone....................................................1 gram.

Sodium Sulfite...................................................5 gram.

Distilled water..................................................100 mls.

Hematoxylin Differentiator: 1% Hydrochloric acid in 70% ethanol

*RESULTS*:

1. Myelin: The myelin should be stained blue or greenish blue

2. Nuclei: The nuclear membrane should be sharply delineated in 
blue-black with the

nucleoplasm clear except for the chromatin, also being blue-black.

3. Cytoplasm and background should be varying shades of red.


NOTE

1. The thickness of the section, the type of hematoxylin, and all the 
times in hematoxylin, eosin, and dehydrating depend upon your requirements.
2. If the section thickness is more than 10 microns, you may want to use 
3 or 4 dips in the Reducer, so as to avoid background myelin staining .
3. The so called "Reducer" (because it is used as a reducer in the 
Bodian silver protein stain) actually functions as a differentiator for 
the myelin stain.