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.



 
Regions within the "stalks" of chromosomes 13,14,15,21, and 22 (the acrocentric chromosomes) contain genes for 18S and 28S rRNA. The silver nitrate stain used in this method binds to proteins near these regions during active transcription. The staining intensity varies from person to person and chromosome to chromosome.

 

Visible are eight of the ten acrocentric chromosomes with their stalk regions staining darkly.

 


NOR-staining (Silver Nucleolar Organizing Region Staining)

This method was successfully used in the Cytogenetics Laboratory of the Laboratory of Pathology in Seattle, Washington, USA.

Principle

Chromosomes are treated with silver nitrate solution which binds to the Nucleolar Organizing Regions (NOR), i.e., the secondary constrictions (stalks) of acrocentric chromosomes.

Equipment

    Incubator set at 37 degrees C;
    30ml capped amber bottle. (Nalgene 2004, VWR #16062-029);
    18 gauge, 1.5" needles. (Monoject 2656);
    1cc tuberculin syringes. (BD 5602);
    02mcg Acrodisc filter. (Gelman #4192, VWR #28144-040);
    12.5cm Whatman #2 Filter paper circles;
    Dish, Petri, Kimax, 150mm X 20mm (VWR # 25353-340;
    Small plastic lids or rings;
    Squirt bottle, filled with distilled water;
    Water bath set at 37 degrees C;
    Glass coplin jar

Reagents

Silver Nitrate (Sigma #S-6506)

Deionized, distilled water

50% silver-nitrate solution: dissolve 5g silver-nitrate in 10ml distilled water and pour into clean and dry amber bottle. Label and store at 4 degrees C.

Procedure

1. Place unstained slide in coplin jar filled with distilled water. Place in 37 degrees C water bath for 2 hours. Remove slide and allow to air dry.

2. Prepare moist chamber by putting two 12.5cm circles of Whatman #2 filter in bottom of glass petri dish. Saturate paper with distilled water. Be sure to remove trapped air bubbles in paper. Place plastic lids or rings on wet filter paper to support each slide at both ends. Cover dish.

3. Attach 18 gauge needle to 1cc syringe.

4. Remove silver-nitrate solution from refrigerator and place bottle in beaker to prevent tipping (see "Precautions" below).

5. Put on gloves.

6. Draw 1cc of silver-nitrate solution into syringe.

7. Remove needle and replace with acrodisc filter.

8. Attach unused 18 gauge needle to acrodisc filter.

9. Lay 5-7 drops of filtered solution near labeled end of slide. Return unused solution to storage bottle. Refrigerate. Discard syringe, filter, and needle in Sharpgard container.

10. Using forceps, lower one end of pre-cleaned coverslip into drops of silver nitrate solution on slide and gently lower coverslip to avoid trapping air bubbles.

11. Transfer treated slide to moist chamber and support on lids above wet filter paper. Cover dish.

12. Carefully place moist chamber in 37 degrees C incubator. Check to see that treated slide is horizontal after closing inner glass door of incubator.

13. Incubate for 7 hours at 37 degrees C.

14. Put gloves back on. Carefully remove slides from moist chamber and using squirt bottle of distilled water wash coverslip and silver-nitrate solution into sink (with tap water running). Rinse slide in coplin jar of distilled water. Let dry. Discard coverslip left in sink using forceps. Do not dismantle moist chamber.

15. Check treated slide under 10X phase and 40X phase to judge effectiveness of first treatment.

16. If stained NOR's are unapparent, repeat steps 3-15 for one hour.

17. Repeat steps 14 and 15.

18. Repeat treatment at on hour intervals two or three times if necessary.

19. Counterstaining is not necessary, but may be disired. Counterstaining with Quinacrine is ideal; however, a plae counterstain with Wright's stain (see "G-Banding" procedure) may work satisfactorily. If Wright's stain is too dark, it is difficult to distinguish between satellites and silver staining on NOR's.

Notes and Precautions

1. 50% silver-nitrate solution should be handled with care. It is a clear solution initially, thus difficult to detect. If allowed to get on skin, it can cause a chemical burn which turns black. This can not be washed off. Discoloration disappears only when a new layer of skin is formed. Clothing is also vulnerable to silver-nitrate staining.

2. Slides that have been previously G-banded are not suitable for NOR staining.

References

Bloom, SE; Goodpasture, C An improved technique for selective silver staining of Nucleolar Organizer Regions in human chromosomes. Human Genetics 34:1990296, 1976.

Lay, YF, Pfeiffer, RA; Arrighi, F.E; Hsa, TC Combination of silver and fluorescent staining for metaphase chromosomes. Am J Hum Genet 30:76-79, 1978.