DE2019778B2 - METHOD FOR DETERMINING THE INTRACELLULAR DISTRIBUTION OF RIBONUCLEIC ACIDS - Google Patents

Description

The present invention relates to a method of structuring orthochromatically, i. H. Light Blue to determine the intracellular distribution of the 25 stain.

Ribonucleic acids by staining previously fixed For testing the method according to the invention

th sections of animal and plant tissue with were mouse and rat liver, rat hepatoma, basic dyes of the thionine group. Larvae of Chironomus tetans and Drosophilla me-

Knowledge of the ribonucleic acid distribution in lanogaster and root tips of different plant ends Cells is of paramount importance to the 30 species used.

Cell biology. To determine the ribonucleic acid, the method according to the invention is described below

numerous processes are already known. ren using an exemplary embodiment in more detail cr-Man knows e.g. metachromatic methods, which purify:

on the property of toluidine blue and the small particles of animal tissues (volume == 1 to

Azurs I and II are based on red-colored compounds 35 2 mm ³ ), larvae or root tips (2 to 3 mm long) with the ribonucleic acid and the fatty substances to be formed with a 12 to 24 hours at 5 to 8 ⁰ C (cf. for example B. Romeis, Mikrosko- the fixing agent given below treats:
pische Technik, 16th edition, Munich 1968, p. 151,

152, 171, 172); However, these methods are unspecific and unselective. The 40 ° ml solution serves the same purpose

Pyronin, which the ribonuclein cell structures red glacial acetic acid 0.4 ml

colors; With this method, however, neither 40% strength formula 1.6 ml is obtained

the detection of the chromosomic ribonuclein Jj _- cobalt nitrate 0.1g

acid, one still gets information about the nucleol uranyl nitrate 0.1 g

structure. 45 50% potassium dichromate solution 3 ml

Furthermore, electron microscopic and auto-ethanol 6 ml

radiographic methods known, in which one ethyl ether 2 ml

Tritiated uridine (H ³ ) as a predecessor in the R-40% formula 4 ml

used bonucleic acid biosynthesis; both methods glacial acetic acid 1 ml

however, require expensive equipment and are 50

difficult to perform. The fixative is applied using ana-

The object of the present invention is therefore to produce lysis-pure substances,
the well-known unspecific staining process with After fixation, the parts are treated with cold water

To improve thionine dyes so that a specific is washed, dehydrated, stored in paraffin and The fischer detection of ribonucleic acids enables cuts of 3 to 5 milliliters according to the usual technique. crown made. After dewaxing, the

According to the invention, this object is divided into two groups A and B,
solves that the sections before staining first with The preparations of group A are 20 minutes

Contains 0.8 percent by volume of concentrated hydrochloric acid - long at 60 ° C, brought into 0.1 N HCl solution, gem Wash methanol and then with 0.5 to 1 weight percent and dehydrate.

centiger aqueous-alcoholic uranyl acetate solution before- Both the preparations treated in this way

are treated and then the staining with group A, as well as those of group B a 0.005 to 0.01 percent by weight, aqueous- are subjected to the same treatment: The alcoholic buffered solution of the dye preparations are used successively in three baths is carried out. 65 brought absolute methanol and then

The method relies on the property of 1 hour at 37 ° C in absolute methanol, which basic dyes from the thionine group and 0.8 ml conc. HCl (D = 1.19) per 100 ml of alcohol but the O-toluidine blue, which contains all ribonucleic substances, was left to stand. Then the

3 - ⁴

■ taken out, at least ten times

exp ^ chilled distilled water at 5 to 8 ° C ^^ ^ ™™ _sub 3uchung of this

washed and subsequently in a 0.5 to l <> / o acetic The ™™ ^ '^ _{zei that} Aue ribonuklei-

uranyl solution entered in SOVoigem methanol and ^ "" ^ t are colored. So appears

Left to stand for one hour at room temperature; 5 ^^ * "J2KRibonic acid in the form of

then the sections are at least ten times with ^. ^^ SSSi red-colored granulations,

washed in chilled water at 5 to 8 ° C and ^^^^^ bon ^ myic acid is identified

fo minutes at room temperature in a buffer ^Οι ^ ° ^ ° ^ _{ε grami} iations running along the

solution of pH 3.4 to 3.6 left standing, the ^^^ th chromosomes are arranged,

is composed as follows: ™ ^^^ i ^ Mie from the giant chromosomes

12.5 ml of the salivary glands voi

8 ml are in the form of

4 ml inclined strips anj

45.5 ml

30 ml schieaener uroncu, ^ m - ——

(The pH is experienced in the solution without methanol ^ o ^^ ^ ndSSmiß can be adjusted advantageously.) _Mm study of Nukleclverhaltens during the

Finally, the preparations are carried directly into the * o ^ «gHf ^ VSS. Ä and freshly prepared dye solution, which through schtose ^ ^ ™ ^^ _{in the cancer} s-dissolving 0.005 to 0.01 weight percent ^^^ processes and can digest O-toluidine blue (Merck) in the above buffer solution ^ _η ^ ° _η ^ _εί5ε the nucleol-organizing was ordered, and lets_45 to 48 hours toZmi- ^ ^ ä ^ ST about the origin of the micro-

give nucleols, nucleolins and karyosomes.

Claims (2)

To stain cell structures on microscopic specimens previously treated with hydrochloric acid containing meth- Patent claims: ethanol and uranyl ions red. 1. Expired to determine the intracellular The specificity of the process is that the ribonucleic acids are dispersed through treatment of the animal or plant tissue staining of previously fixed sections of animal sections with hydrochloric acid-containing methanol, the group and plant tissues with basic colorations - COOH (through methylation) and substances of the thionine group, thereby identifying - O - SO ₃ H (through methanolysis) of the acidic polycyzine. that the sections are blocked saccharide prior to staining, which prevents them from taking part in metachromatic reactions with methanol containing acids and then with. 0.5 to 1 percent by weight aqueous-alcoholic By using the dye in aqueous Uranyl acetate solution will be pretreated and an alcoholic solution will continue to be decisive of the danger the staining with a 0.005 to 0.01 countered that the acidic cellular fat substances metachromatically percent by weight aqueous-alcoholic buffered 15 react, since even the smallest amounts of alcohol the A wide solution of the dye is carried out. metachromasia formed by these fatty substances 2. The method according to claim 1, characterized by none. By using the uranyl acetate solution indicates that, as a basic dye, the becomes under the working conditions of this process Thionine group O-toluidine blue is used. ensures that only cell ribonucleic acid ₂₀ because of the phosphate contained in the molecule group - persistent metachromatic complexes d. H. forms red-colored complexes with the dye, while the other non-ribonuclein cells