Method for staining nucleolar organisers on histological preparations and cytological smears


G01N1/30 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

FIELD: medicine.

SUBSTANCE: paraffin sections are prepared, fixed, coated with a colloidal developer prepared by mixing 2% aqueous gelatin in 1% formic acid and 50% aqueous AgNO3 in equal proportions, incubated for 30-60 minutes, washed and contrasted. The material is fixed in 10% neutral buffered formalin (pH 7.4) for 24 hours. The material is finished in ascending alcohols, encapsulated in paraffin to prepare the sections which are prepared in 2% formic acid in 96% ethanol for 20 minutes, dehydrated in 2 portions of 96%; the sections are prepared in 0.1% NaOH for 2 min 30 sec. Then they are dried. One drop of 100% AgNO3 is layered on the preparation. It is followed by incubation in a thermostat at 60°C for 1 min 40 sec in a humid chamber, and coating with one drop of 40% formaldehyde and colloidal developer. It is incubated for 20-50 seconds in the thermostat at the same temperature. The sections are washed in 4 portions of distilled water. The sections are processed with acetate buffer pH 2.4 for 1 minute; the preparations are contrasted with 0.2% aqueous methyl green for 10-15 seconds, purified in chloroform, differentiated for 2-3 minutes in n-butyl alcohol, processed in toluene and encapsulated in polystyrene.

EFFECT: higher quality of detection and evaluation of nucleolar organisers.

1 ex, 2 dwg

 

The invention relates to medicine, namely to the pathology, cytogenetics, and can also be used in various fields of biology, embryology, Cytology and histology.

Is currently the urgent problem of differential diagnosis of precancerous diseases and tumor growth, as well as prediction of malignant tumors. One of the ways to solve the problem, is the method of staining nucleolar organizers.

The following analogues of the method of staining nucleolar organizers.

The method of staining in C. Goodpasture, Bloom S.E. (C. Goodpasture, S.E. Bloom // Chromosoma. - 1975. - Vol.53. - R-50): 800 mg of silver nitrate (AgNO3) dissolved in 2 ml of acetate buffer Walpole (0.1 M CH3COONa and 0.1 M CH3COOH in the ratio 2:18) at pH 3.7 and titrate with 25% ammonia solution until the disappearance of turbidity. Immediately before staining drugs mix three drops of the solution prepared in AgNOR3with one drop of a 3% solution of neutral formalin (pH 7.0). One or two drops of the mixture applied to the preparation, cover with a cover slip and incubated for 5 to 45 min at +55 to +65°C. the incubation Time drug pick empirically; the painting quality is controlled under the microscope.

The staining method is based on the binding of bismuth ions:

1. Prepare in the usual way the m paraffin sections of a thickness of 3 μm from a fixed neutral formalin tissue, deparaffinized them and registryroot in pure water.

2. Wash sections in 0.2 M Tris - HCl, pH 7,6.

3. Stained in a solution of bismuth for 2.5 hours or more.

4. Washed three times for 10 min in 0.1 M Tris - HCl, pH 7,6.

5. In a fume hood washed slices of 0.1 M Tris - HCl, pH 7,6, to which are added a few drops of ammonium sulfide.

6. Wash the slices in pure water, digitalout and tear under the polystyrene. Nucleolar organizers look like black and brown dots.

The disadvantages of the known methods are:

1. The complexity of the preparation of solutions.

2. The duration of the painting.

3. Coverage of the chromatin of the nucleus, which affects the calculation of nucleolar organizers.

4. Precipitation of metallic silver.

5. The peeling of the cuts from the glass during the painting.

6. The use of expensive and scarce reagents.

7. The use of toxic ammonium sulfide.

The closest achieved technical result is a method for staining of nucleolar organizers in W.M. Howell, D.A. Black (W.M. Howell, D.A. Black // Experientia. - 1980 - Vol.36. - P.1014-1015).

The method is as follows.

1. The usual method of preparing paraffin sections of a thickness of 3 μm frozen sections with a thickness of 4 μm or PAP smears. Preparations fixed in 100% ethanol or 100% methanol at room temperature for 10 minutes.

2. Mix equally in the x quantities of the following chemicals:

Gelatin 2% aqueous solution of formic acid 1% and 50% aqueous solution of AgNO3.

3. Pour the mixture slices and incubated them for 30-60 minutes in a dark place at room temperature. Thoroughly washed drugs in double-distilled and deionized water.

4. If necessary, conduct the contrast in the standard solution hamalawy Mayer in 2-5 minutes

5. Dehydration slices in the battery alcohols (for example, 50, 70 and 100% ethanol), illuminate in xylene and mounted using synthetic environment.

The disadvantages of this method are:

1. The difficulty of obtaining deionized water.

2. Akrasanee chromatin of the nucleus.

3. Not always stable results of color.

4. Precipitation of metallic silver.

The authors propose the method of staining of nucleolar organizers in histological sections and cytological smears, allowing high quality to assess the activity of nucleolar organizers that can improve differential diagnosis and prognosis of malignant tumors.

The technical result of the claimed process is raising awareness identify nucleolar organizers.

The figure 1 presents the results of staining of nucleolar organizers in cancer of the colon is iski (from 10 to 18 even-numbered dots in the nuclei of cells). Histological preparation. The magnification ×1000.

The figure 2 presents the results of staining of nucleolar organizers in cancer of the stomach. A large number of distinct black dots in the nuclei of cells. Cytological preparations. The magnification ×1000.

The proposed method has in common with the prototype of signs, namely: prepare paraffin sections, fix, put colloidal developer obtained by mixing equal amount of 2% aqueous solution of gelatin in 1% formic acid and 50% water solution of AgNO3and incubated for 30-60 minutes, washed in distilled water, in contrast.

The difference between the claimed method from the prototype is that the fixation of the material is carried out in a 10% solution of neutral formalin, buffered by Lilly (pH 7.4), within 24 hours, the wiring material is carried out in alcohols of increasing concentration, pour in paraffin and prepared slices treated at 4°C in fluid Carnoy (1 part glacial acetic acid and 3 parts of 96° ethyl alcohol), then treated slices 2 N. solution of formic acid at 96° ethyl alcohol for 20 minutes, washed, sliced in two portions 96° ethyl alcohol, handle sections in 0.1 G. of NaOH, prepared at 500° ethanol for 2 min 30 s, dried slices on the air layer on the product one drop of 100% rest the RA AgNO 3and incubated in a thermostat at 60°C for 1 min 40 s in a humid chamber (Petri dish with moistened filter), then put 1 drop of 40% formaldehyde solution and 1 drop of colloidal developer, incubated for 20 to 50 with thermostat at the same temperature, rinse the slices into 4 portions of distilled water, then treated slices in acetate buffer (pH 2,4) for 1 minute, contrast preparations of 0.2% aqueous solution of methyl green for 10-15 seconds, differentiate 2-3 minutes in n-butyl alcohol, illuminate toluene and sign in polystyrene.

Result: the background is painted in a greenish color karyoplasm defined light brown nucleoli, which are clearly visible granules of black color.

Staining of cytological preparations is carried out in the same way.

Thus, the advantages of the presented method compared with the existing variants of the method of detection of nucleolar organizers are: the absence of expensive reagents, speed painting, stability and precision of detection of silver grains in the nucleoli, no precipitation of metallic silver, reducing paint chromatin of the nucleus, in addition even thick histological sections do not come unstuck from the slide during staining. When applying this method of akrasian what I'm on cytological smears do not wash material including tissues that are rich in mucilage (stomach, endometrium, cervix and other) due to the fact that formic acid, prepared in ethanol, which is a lock for mucus. The claimed method is effective and technically easy to implement in any morphological laboratory and pathology Department.

The method of staining nucleolar organizers on histological preparations and cytological smears, lies in the fact that prepare paraffin sections, fix, put colloidal developer obtained by mixing in an equal amount of gelatin 2%in aqueous solution at 1% formic acid and 50%water solution of AgNO3, incubated for 30-60 min, washed and contrast, characterized in that the fixation is carried out in a 10%solution of neutral formalin buffered by Lily (pH 7.4) for 24 h, the wiring material is carried out in alcohols of increasing concentration, pour in paraffin and prepared sections, which is treated in 2 N. solution of formic acid at 96° ethyl alcohol for 20 min, the dehydration is carried out in two portions 96° ethyl alcohol, treated slices in 0.1 G. of NaOH for 2 min 30 s, dried, layer on the drug one drop 100%-aqueous solution of AgNO3then incubated in a thermostat at 60°C for 1 min 40 s in humid chambers is, put 1 drop of 40%formaldehyde solution and colloidal developer, incubated for 20 to 50 with thermostat at the same temperature, washed slices into 4 portions of distilled water, treated slices acetate buffer pH 2.4 V for 1 min, contrast preparations of 0.2%aqueous solution of methyl green for 10-15 s, distilled chloroform, differentiate 2-3 min in n-butyl alcohol, treated with toluene and conclude in polystyrene.



 

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