APPENDIX 4

REAGENTS

Biuret Reagent

In 500 ml distilled or deionized water, dissolve:

Copper sulfate (CuSO4.5H2O) 1.5 g

Sodium potassium tartrate 6.0 g

(NaKC4H4O6.4H2O)

To this mixture, add 300 ml of CO2-free 10% NaOH slowly under

continuous stirring.

Add CO2-free H2O to make this reagent up to 1 l and store in a

tightly screw capped polyethylene or glass bottle in the cold.

Nessler's Reagent

In 15 ml of distilled water, dissolve:

Mercuric chloride 1.0 g

Potassium bromide 5.0 g

Sodium hydroxide 2.5 g

Dilute to 100 ml, refrigerate. Allow to sit in the refrigerator

for 5 days. Use the upper clear solution only, or filter.

Preparation of Gelatin-Rhodamine Isothiocyanate (RhITC) Conjugate

1. Prepare a 2% gelatin solution.

2. Add 1N NaOH dropwise until pH reaches 10-11.

3. Autoclave for 10 min at 15 psi and 121C.

4. After cooling add rhodamine isothiocyanate (RhITC) dissolved

in a minimum volume of acetone to provide 8 μg of dye per

one mg of gelatin. Remove residues by filtration through a

45μm membrane filter.

5. Allow conjugation to proceed overnight with gentle stirring.

6. The conjugate is separated from unreacted RhITC by gel

filtration on Sephadex G-25, using PBS pH 7.1 (alternatively

the preparation could be dialyzed against PBS pH 7.1 until

no further color is detected in the dialysate).

7. Add merthiolate to the conjugate (1:10,000) and distribute

the conjugate in small volumes into screw-cap tubes and

store at -20C. Alternatively, the bulk of the conjugate

could be freeze-dried and stored in a desiccator. When

needed, the desired amount of the dry sample should be

reconstituted in distilled water.

Mounting Medium (taken from Kawamura, 1969)

Buffered glycerol or elvanol is commonly employed. Fluorescence

fades in a short time (about 30% overnight and then more

gradually) in glycerol but remains for a longer time in elvanol.

The fluorochrome of the rhodamine series dissolves in elvanol,

however, and therefore it cannot be used except with FITC-labeled

antiserum. The pH of the buffered glycerol is normally 7.0 to

7.5. However, we have used it at a pH of 8.5 with good results.

Buffered glycerol solution

0.5 M carbonate buffer (pH 9.5) l vol.

Glycerine (reagent grade, 9 vol.

free of autofluorescence)

The two reagents are mixed thoroughly (with a magnetic stirrer).

The final pH should be 8.5.

Elvanol (Elvanol-buffered glycerine mixture)

Elvanol (polyvinyl alcohol, 51-05 grade) 1 vol.

0.5 M carbonate buffer (pH 9.0) 4 vol.

The two reagents are mixed with a magnetic stirrer for 16 h. One

volume of reagent grade glycerine is mixed with two volumes of

the above mixture. The final mixture is stirred again with a

magnetic stirrer for 16 h, centrifuged for 60 min at 12,000 rpm

and the pH of the supernatant corrected to 8.5.

The final product should be kept in an air-tight container. It

is best stored in tubes and kept in the dark. It will harden

under the cover glass and fix it firmly.