Specimen preparation and observation experiments of silver-stained nucleolus-forming region
Specimen preparation and observation experiments of silver-stained nucleolus-forming region
In recent years, it has been found that although the short arm of the human telomeric chromosome is the site of the 28S, 18S, and 5.8S rRNA genes, called the nucleolus-forming region, the silver-stained material is not rDNA and rRNA, but a class of acidic proteins related to active rDNA transcription, which can easily reduce Ag ions to particles of Ag, and thus silver plated and brownish-black in the nucleolus-forming region, which is known as the silver-stained nucleolus-forming region.
Operation method
basic program
Principle
Biochemical and immunochemical studies have demonstrated that silver-stained proteins are RNA polymerase I, whose function is to catalyze the transcription of rRNA from rDNA to form nucleoli, and thus are able to specifically display rRNA transcriptional activity by this reaction.
Materials and Instruments
HL-60 cells HeLa cells Move I. Silver staining and observation of the nucleolus-forming region of intermediate chromosomes For more product details, please visit Aladdin Scientific website.
Carnoy's Fixative HCl Formic Acid AgNO3 Protein Gel Sodium Thiosulfate Glutaraldehyde Ethanol Acetone Uranyl Acetate Acetate Lead Citrate Staining Solution
Microscope Electron microscope Ultrathin slicer Water bath Centrifuge Balance Staining vat Flat dish Nipple pipette Centrifuge tube Mirror paper
1. Take the prepared chromosome specimens of normal or tumor cells and put them directly into the solution containing 5 N HCl and treat them for 5 minutes at room temperature.
2. Rinse several times with tap water, shake dry, and place the specimen face up in a flat dish.
3. Place 0.1% formic acid in 50% AgNO solution, about 0.5 ml, on the specimen with a pipette and cover with two pieces of microscope paper.
4. Place the petri dish in a 56 ℃ water bath for 3 ~ 5 minutes, until the microscope paper is brown, remove the specimen rinsed with water, air dry.
5. Mirroring
(1) The background of the specimen under the microscope is light yellow, the karyotype is deeply stained, and the silver-stained proteins of the end-stranded chromosomes are present in brownish-black particles. Select the split phase with well-dispersed chromosomes and clear silver-stained particles, and count the number of chromosomes that have silver-stained particles unilaterally or bilaterally.
(2) The silver-stained particles in human cells are genetically stable, with a normal value of 4~8/karyotype.(3) Calculation method: NOR average = the sum of the number of NOR chromosomes in N karyotypes / N karyotypes.
Second, the interphase cell nucleus active nucleolus formation area of silver staining and observation
1. Collect cultured HL-60 cells and human normal peripheral blood lymphocytes transformed with PHA in centrifuge tubes, centrifuge at 1,000 rpm for 5 minutes and discard the supernatant.
2. Suspend the cells by pipetting or finger-flicking, and then fix them with Carnoy's fixative for 30 min.
3. Centrifuge the cells at 1,000 rpm for 5 minutes, discard the supernatant and leave about 0.5-0.1 ml of liquid, suspend the cells and titrate the slides.
4. Dry at 37℃ for 24 hours.
5. Silver staining
6. Microscopic examination
(1) Under the microscope, the nucleus is colored but the cytoplasm is not, and the silver-stained particles in the nucleus of the active nucleolus-forming region are brownish-black, clustered and clearly distinguishable.
(2) There are obvious differences in the number and appearance of silver-stained particles in tumor cells and normal cells.
Preparation and observation of electron microscope specimen of silver-stained active kernel-forming region in interphase cell nuclei.
1. Collect cultured HL-60 cells or HeLa cells in a centrifuge tube, and then add 2.5% glutaraldehyde 1 ml to pre-fix it for 10 min.
2. Centrifuge at 1,000 rpm for 10 minutes, discard the supernatant and add Carnoy's fixative for 5 minutes.
3. Next, centrifuge the cells at 2,500 rpm for 10 minutes, discard the supernatant and blot the residual liquid with absorbent paper, and remove the cell pellet with an ear spoon and place it in a small flat dish.
4. 100% ethanol→double-distilled water gradient rehydration for 5 min each step.
5. The cell mass was then cut into pieces of approximately 1 mm3 in size.
6. 2~3 ml of a 2:1 mixture of 50% AgNO3 (prepared with distilled water)-2% protein gum (prepared with 1% formic acid) was added to a small flat dish with cell mass blocks.
7. Remove the petri dishes and treat in a 56°C water bath for 20 min.
8. Thoroughly wash with water 3 times for 2~3 minutes each time.
9. Treat with 5% sodium thiosulfate for 10 minutes at room temperature after water washing.
10. Wash with water 3 more times and transfer to ethanol-acetone gradient dehydration for 5 minutes per step.
11. Epon812 embedding.
12. Ultrabook sections were visualized under an electron microscope either directly or after re-staining with uranyl acetate and lead citrate.