Silver Nitrate staining of Nuclear Organizing Region (NOR) Introduction The nucleolus organizer region (NOR) or nucleolar organizer

is a chromosomal region around which the nucleolus forms and contains several tandem copies of ribosomal RNA genes for 5.8S, 18S, and 28S rRNA. These are clustered on the stalks / satellites present on the short arms of acrocentric chromosomes 13, 14, 15, 21 and 22. Silver staining can be used to identify the NOR, as, Silver nitrate inserts into the NOR-associated protein and stains them dark black. Interphase nuclei show intense dark brown staining of the nucleolus. Ideally, a metaphase should have a maximum of 10 NOR sites. However, due to variations in distribution and activity of the ribosomal genes (NOR), only 5 to 10 NOR are stained. Materials required: Cultured peripheral blood lymphocytes (fixed in Carnoys fixative) Microscopic glass slides Gelatin solution, 2% (w/v) Silver nitrate solution, 50% (w/v) Giemsa stain

Preparation of reagents: Gelatin solution, 2% (w/v) Formic acid - 1 ml Gelatin - 2 g H2O - 99 ml The mixture was stirred with heat to get gelatin into solution. After complete dissolution, the colloid can be stored in a dark bottle at room temperature for <1 year. Silver nitrate solution, 50% (w/v) Silver nitrate crystals - 5 g (Silver nitrate crystals should appear white, not gray) H2O - 10 ml The solution can be stored in a dark bottle for <1 year at 4C Note:

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. Procedure: Freshly prepared slides with good number of well spread metaphases were selected. Using a pasteur pipette, 3 drops of gelatin colloid solution were mixed with 11 drops of silver nitrate solution. 4-6 drops of the mixture were placed on the slide and covered with a coverslip Excess liquid was blotted with tissue paper. (This is done to minimize background of silver and
gelatin grains)

The slides were incubated on a slide warmer at 50C for 4 - 6 minutes depending on the formation of a yellow color precipitate. (If color turns brown the slide is usually over-treated). The coverslip was removed from the slide by rinsing in dH2O. The slides were stained for for 2 - 4 minutes with 4% Giemsa stain in Sorensen buffer. After rinsing with cold water and air drying, the slides were ready for analysis.

Procedure 2: Onto the test slides, two drops of 1% aqueous gelatin solution with 0.25% formic acid and four drops of silver nitrate at 25% were placed. The slides were covered with coverslips and incubated for five seconds in the presence of high-potency microwaves. After incubation, the coverslips were removed and the slides were washed under tap water, stained with 5% Giemsa for 30 s, and airdried. Observation Light golden-brown stained chromosomes with dense brown staining at nucleolar organizer regions were observed. Add a point on how many NOR were seen per metaphase. Discussion Variations in NOR staining are inherited within families and this technique is useful for analysing chromosomes with double satellites, chromosome polymorphisms and structural abnormalities involving satellite regions. The number of NOR in a cell also rises with increasing proliferative activity of cells. Hence, silver nitrate staining for NOR is helpful in the delineation of malignant tissue from normal or benign lesions. 2