[Histonet] How to stain mast cell in mouse lung sections with toluidine blue?

[min he]

Dear all,

I am using toluidine blue to stain mast cell in mouse lung tissue sections
for several times. The results are frustrating. The background of my
staining is blue and the purple staining of mast cell was not observed in
several sections. I use mouse ear sections as a positive control. The
staining of mast cell in ear sections are much better: mast cell-purple,
background blue. Would you please share your experience on mast cell
staining with toluidine blue? Could it be the problem of fixation? I put the
lung samples in formalin for several days before processing them.

The following is the protocol I am using:
**

Acidified toluidine blue
*SPECIMEN PREPARATION
*Cut 3 to 5 µm thick paraffin sections from tissue fixed in 10% neutral
buffered formalin. Tissue known to contain mast cells (neurofibroma, skin)
is used as a control.
*

REAGENT PREPARATION
*1 *0.5% aqueous potassium permanganate
*Potassium permanganate 0.5 g
Distilled water 100 ml

2 *2% aqueous potassium metabisulphite
*Potassium metabisulphite 2 g
Distilled water 100 ml

3 *Acidified toluidine blue solution (pH 3.2)
*Distilled water 99.75 ml
Glacial acetic acid 0.25 ml
Toluidine Blue (CI 52040) 0.02 g
*

METHOD
*1 Dewax and rehydrate sections.
2 Transfer sections to potassium permanganate solution for 2 minutes.
3 Rinse in distilled water.
4 Transfer sections to potassium metabisulphite solution for 1 minute (or
until sections appear white).
5 Wash in tap water for 3 minutes.
6 Rinse in distilled water.
7 Place in acidified toluidine blue solution for 5 minutes.
8 Rinse in distilled water.
9 Dehydrate rapidly, clear and mount.
Thank you very much.

Best wishes,

He Min