Feulgen Cytophotometery

E.S.Cole 2005

 

Slide Preparation

 

Preparation of reagents

 

1) Prepare a series of clean Coplin jars. (Use nitric acid to remove pre-existing stain if

            necessary).  13 with ordinary lids and 2 with glass lids.

 

2) Locate or prepare reagents:

 

            Carnoy’s Fixative:

                        3:1 ETOH: Acetic Acid

                        add 75 mLs 95% non-denatured ETOH to 25 mLs glacial acetic acid.

 

            ddH2O rinse

 

            5M HCL (glass top Coplin jar)

 

            ddH2O Rinse

 

            Schiff’s Reagent*  (See special instructions for preparation.)

 

            ddH2O

 

            “Bleach”  (must be made up fresh the day of use!)

                        18 parts dH2O  (180 mLs)

                        1 part 1 M HCl  (10 mLs)

                        1 part 10% Potassium metabisulfite (Make up fresh!)

                             (weigh out 1 g, PMBS) place in capped 15 mL centrifuge tube.

                             Measure out 10 mLs of H2O in a second 15 mL centrifuge tube.

                             When ready to use, mix water and powder, and then add to H2O and HCl.

 

            2 X ddH2O rinse.

 

            Ethanol series:  30%, 50%, 70%, 95%, 100%, 100%.

 

            Find Euparal vert mounting medium.

 

3) Set up staining station in Hood (Use gloves and lab jacket).

 

4) Stain:   Fix 5 min in Carnoy’s (unless slides not fixed, then 15 min). 

            Rinse in dH2O. 

            Transfer slides to acid for hydrolysis: 20 min.  (Time is critical).  Rinse.

            Transfer to Schiff’s (should be clear) 2hr room temp. 

            Bleach 10 min.  Rinse 2 X.       

            Dehyderate in ETOH series (2-5 min each).  Air dry slides. 

            Add 3 small drops Euparal vert, and long coverslip.  Allow to dry overnight. 

            Seal edge with mounting medium or nail polish.

Schiff’s Reagent Special Preparation.

 

1.  Dissolve 1 g basic fuchsin in 200 mL boiling distilled water, slowly and carefully.

 

2.   Shake well.

 

3.  Cool to 50 C.

 

4.  Filter and add 30 mL 1 M HCl to the filtrate.

 

5.  Add 3 g potassium metabisulfite in Hood.

 

6.  Bleach overnight in tightly stoppered bottle in dark at room temp.

 

7.  Add 0.5 g  activated charcoal.

 

8.  Filter reagent.

 

9.  Store bottle in refrigerator.  Lasts in refrigerator up to six months or more.