SILVER STAINING OF NUCLEOLAR ORGANIZING REGIONS (NOR)

I. Purpose:

A. To locate nucleolar organizer regions on chromosomes. Useful for studies of chromosomes with double satellites, chromosome polymorphisms, and structural abnormalities involving satellite regions.

II. Procedure:

A. Harvest cell culture according to the standard procedure. Allow the slides to dry at room temperature. After the slides have dried completely, they can be stained at any time.

B. Wear gloves during procedure and protect work area with lab mat or paper towels since silver nitrate is a hazardous chemical that may cause burns, In addition, if it contacts the skin it will stain so un-noticed spills can cause problems for other workers.

C. Using pasteur pipet, measure 3 drops of gelatin colloid solution. With a fresh pipet add 11 drops of silver nitrate working solution. Working quickly, mix by pipeting up all liquid and then place 4 drops of mixture on the slide. Cover with a 24 x 60 mm coverslip and blot between paper towels to remove excess liquid. This is done to minimize background of silver and gelatin grains.

D. Incubate slide on slide warmer at 50 C for 4 - 6 minutes. Slide will turn yellow color, if color turns brown the slide is usually over-treated.

E. Remove coverslip by rinsing in dH2O.

F. Stain slides for 2 - 4 minutes with Leishman stain in Gurr buffer (1:3, prepared fresh). Rinse with cold water, air dry. Slides are now ready for analysis.

III. Solutions:

Gelatin colliod: 2% powdered gelatin, 1% pure Formic acid in dH2O. Generally we prepare 2 - 5 ml of solution at a time.

Gurr Buffer: Dissolve 1 Gurr buffer tablet pH 6.8 (1 liter solution) in 1 liter dH2O.

Leishman Stain: Dissolve 1 gram Leishman stain in 500 ml Methanol. Incubate at 37 C overnight. Filter through Whatman paper qualitative grade 1.

Silver Nitrate: 50% Silver Nitrate dissolved in dH2O. Sterile filter through a 0.22 u filter. Store in brown bottle or by other light-protective means.

IV. Reagents:

Formic Acid: 91.5% Formic Acid.

Gelatin powder:

Gurr Buffer Tablets: BIO/MEDICAL SPECIALTIES cat# 33193-2P. Buffer Tablets pH 6.8 (1 liter of solution) BPS 50 tablets/bottle.

Leishman Stain: AMERICAN SCIENTIFIC PRODUCTS cat# S7710-27. Harleco Leishman Stain, 25 gram/bottle. Store in dark.

Methanol: AMERICAN SCIENTIFIC PRODUCTS cat#3016-1. Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free, 500 ml bottle.

Silver Nitrate: FISHER SCIENTIFIC CO. cat# S-181. Silver Nitrate, crystal, ACS certified, 25 gram/bottle. Store in dark.


Reference: Gold, J.R., Ellison, J.R.:Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes. Stain Tech. 58: 51, 1983.