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 CO2free 10% NaOH slowly under continuous stirring.

Add CO2free H2O to make this reagent up to 1 l and store in a tightly screw capped polyethylene or glass bottle in the cold.

Nessler'sReagent

In 15 ml of distilled water, dissolve:

Mercuric chloride1.0 g

Potassium bromide5.0 g

Sodium hydroxide2.5 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 GelatinRhodamine Isothiocyanate (RhITC) Conjugate

1.Prepare a 2% gelatin solution.

2.Add 1N NaOH dropwise until pH reaches 1011.

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 G25, 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 screwcap 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 FITClabeled 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 (Elvanolbuffered glycerine mixture)

Elvanol (polyvinyl alcohol, 5105 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 airtight container. It is best stored in tubes and kept in the dark. It will harden under the cover glass and fix it firmly.