Crowle's stain
Crowle's stain - for immunodiffusion
250 mg Crocein Scarlet MOO, 3B
15 mg Coomassie Brilliant Blue R250
100 ml 5% acetic acid, 3% trichloroacetic acid
Stir at 60oC until dissolved, filter (Whatman no.1). Filter after each use.

Staining procedure

1- Wash gel 2 hrs each with two changes of buffer (phosphate or Tris buffered saline). For silver staining, gels require more exhaustive washing.
2- Wash 1 hr with distilled water.
3- Place slide, gel side up, on filter paper. Cover with 2 (or more) pieces of filter paper. Cover with 2X folded paper towels.
4- Press assembly with an inverted Petri dish lid containing a beaker with about 500 ml of water.
5- After 30-45 min, remove the gel and air dry.
6- Stain 30 min (or more).
7- Wash and soak about 30 min in 0.3% acetic acid.
1- Agarose adheres better to GelBond than to glass.
2- If you use microscope slides, make sure they're clean.
3- Coomassie Blue is relatively insoluble. If proteins stain red instead of blue, heat the stain to dissolve the dye, or replenish the Coomassie Blue.
4- If gels dry before washing, protein will denature giving a high background.
5- Stained gels may be stored indefinitely.
6- For electrophoresis gels the exhaustive washing is unnecessary
7- The residual charge of agarose leads to high background with standard polyacrylamide staining procedures
8- Crocein scarlet is purely anionic and hence has little tendency for background staining
9- The basic procedure is from J. Immunol. Meth. 17, 379-381 (1977).