4.4. Eriochrome Cyanine Staining

Eriochrome cyanine staining (EC)

Eriochrome cyanine (EC) staining is a commonly used technique for the analysis of CNS demyelination (Moliné-Velázquez et al., 2011; Moliné-Velázquez et al., 2014).

1-Briefly, the hystological sections were RT-dried during 1-2 hours

2-Drying the slides on strove at 37º C , 1-2h

3- Exposed to acetone 5 minutes. Place the slides in baskets and in the bucket with acetone in the flow hood. 5 minute a RT.

4-Drying the slides in the flow hood, to RT 30 minutes

5-Filter the solution Eriochrome cyanine and submerged into the staining solution containing 0,2% Eriochrome Cyanine , 0.5% sulfuric  acid  and 4% iron aluminum . (It can be kept up to 6 months, depending on the times it is used).

6-Put the slides in the solution Eriochrome cyanine 30 minutes to RT

7-Wash the slides with tap water several times to remove excess dye. Wash with destilled water last time

(Wash 3 times for 10 minutes each)

8-Differentiatte 5 % iron aluminum for 10 minutes to RT (The gray matter will look gray and the white matter will look blue)

9--Wash the slides with tap water several times to remove excess dye. Wash with destilled water last time

(Wash 3 times for 10 minutes each)

10-Filter the borax solution.

11- Were exposed to a borax solution for 2-10 minutes to RT .We must look at the microscope and check that the tissue is well differentiated. The gray matter should be white and the white matter should look blue. Wash the tissue with borax until this is differentiated.

12--Wash the slides with tap water several times to remove excess dye. Wash with destilled water last time

(Wash 3 times for 10 minutes each)

13- Dehydration. Place the horizontal buckets and change the baskets

ethanol  70º. 10 minute

b. ethanol  80º. 10 minute c. ethano l 90º. 10 minute d. ethanol 100º. 10 minute e. ethanol 100º. 10 minute in different basket. f. xylol.10 minute. g Xylol 10 minutes in different basket.
(during the xylol, if it gets cloudy is that it has a lot of water and you have to start over the dehydration)

14- DPX new: non aqueous mounting medium for microscopy.  (contains m- xylene)
place coverslips on top of the slides with DPX

SOLUTIONS

10% IRON ALUMN (Ferric Ammonium sulfate)

Sigma:  F1668  

MW: 482.19

Add 10Gr of ferric ammonium sulfate in 100 Ml of distilled water.

Shake until the solution is complete.

Protect from light.

Filter before using

5% IRON ALUMN (Ferric Ammonium sulfate)

Sigma:  F1668.  

 MW: 482,19

Add 5Gr of ferric ammonium sulfate in 100 Ml of distilled water.

Shake until the solution is complete.

Protect from light.

Filter before using

ERIOCHRME CYANINE SOLUTION

Millipore: C.I 43820

Weigh 0.2 grams of Eriochrome cyanine in a beaker and add 0.5 milliliters of concentrated sulfuric acid. 

Add 96 milliliters of distilled water and stir until the solution is colored. 

Add the solution of 10% iron aluminum, mix well and protect from light

BORAX-FERRICYANIDE DIDFFERENTIATOR

BORAX: SODIUM TETRABORATE DECAHYDRATE.SIGMAULTRA, 99.5-105.0%

SIGMA ALDRIGH: B3545-500G

MW: 381.37

Across organics: Potassium ferricyanide 99+% for analysis 

MW: 329.26

Weigh 1Gr of borax and 1.25 grams of potassium ferricynide in 100 milliliters of distilled water. Agitate to dissolve .Protect from the light