I am doing a stain for myelin using Eriochrome Cyanine (Solochrome Cyanine). The counterstain is a neurofilament stain visualized using DAB. I am working from 45 micron sections of brain tissue. After the DAB step to finish the neurofilament stain, I wash in distilled water, then incubate for 8 minutes in acetone. After the acetone, I do 4 rinses with distilled water for 3 minutes each time, after which I stain in Eriochrome Cyanine for 30 minutes.
The last few times I have run the protocol, there have been patches of tissue where the Eriochrome Cyanine dye does not impregnate; these patches on the tissue appear white instead of dark purple/blue like the stained sections. This issue persists through the rest of the protocol, and there are parts of the tissue on the finished slides where myelin is definitely present but was not stained blue (i.e. the corpus callosum).
Has anyone had this issue? I am thinking maybe there is water preventing the stain from impregnating or something similar. Thanks.
Hi Elyse,
what did you do after 30 min incubation with EC?
Please check this protocol as well.
EC Staining Protocol
1.Take the slides out of the freezer and allow to air dry for a couple hours or overnight at room
temperature, or dry the slides on a slide warmer for 1-2 hours at 37c.
2.Place slides in fresh Acetone for 5 minutes.
3.Remove slides from Acetone and let dry for about 10-20 minutes.
4.Place the slides in Eriochrome Cyanine Solution for 30 minutes at room temperateure. Note:The solution was reusable, filter the Eriochrome Cyanine Solution if needed.
5.Rinse the slides with running water.
6.Differentiate in 5% Iron Alum for 5-15 minutes until start to see the gray matter.
(Usually about 2-5 min for rat spinal cord and 5-10 min for mouse tissue. Do not reuse this solution.) 7.Rinse with running tap water.
8.Complete the differentiation in the borax-ferricyanide solution (reusable) for 5-10 minutes. 9.Rinse with running tap water.
10.Dehydrate briefly through graded ethanol solutions 2 changes each, 70%, 95% and 100%.) 11.Clear briefly through 3 changes of Histoclear.
12.Coverslip using a permanent mouonting medium.
Results:
Myelin: blue
Nuclei: cream/white
Background: cream/white
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