RU2198404C2 - Method for detecting alkaline phosphatase in immunocompetent organs in animals - Google Patents

Abstract

FIELD: veterinary medicine. SUBSTANCE: method deals with immunology of agricultural animals and poultry. At studying histological sections to prepare incubation medium one should use sodium teraborate taken at 1:1 ratio with naphthylphosphate. The method provides decreased economical expenses. EFFECT: simplification of process.

Description

 The invention relates to veterinary medicine, in particular immunology of farm animals and birds.

 A known method for the determination of alkaline phosphatase (see 3. Loyd "Histochemistry of enzymes". M.: Mir, 1982. Simultaneous a combination according to Burstone 1962, p. 64), including tissue preparation, fixation, incubation with subsequent staining and accounting for the results.

 The closest is a method for determining the activity of alkaline phosphatase (see 3. Loyd's "Histochemistry of Enzymes". M: Mir, 1982. Simultaneous azo matching according to Pearse 1953, p. 63), which provides for the study of histosections, by preparing tissues, taking, processing, fixation, washing and filling of the sample, its incubation in a medium consisting of a buffer solution, distilled water, naphthyl phosphate and blue durable B, washing and additional staining.

 The disadvantage of this method is the complexity of the preparation of veronal acetate buffer or Tris-HCl, the use of expensive components.

 The technical solution to the problem is to simplify the research process and reduce economic costs.

This object is achieved in that the method for detecting alkaline phosphatase in the immunocompetent organs of animals involves the study of histosections by preparing tissues, taking, processing, fixing, washing the sample, incubating it in a medium consisting of buffer solution, distilled water, naphthyl phosphate and a neutral strong blue dye In, washing additional staining. The quality reaction to alkaline phosphatase is assessed by color; sodium tetraborate Na ₂ B ₄ O ₇ • 10H ₂ O, which is taken in the ratio with naphthyl phosphate 1: 1, is used to prepare the buffer solution.

 The novelty of the proposed proposal is due to the fact that the use of sodium tetraborate together with naphthylphosphate provides an effective incubation of histosections by creating a certain pH leading to the activation of alkaline phosphatase.

 An example of a specific implementation of the method for determining the activity of alkaline phosphatase in the immunocompetent organs of an animal.

 Alkaline phosphatase enzyme code (3.1.3.1.) Refers to the II group of the enzyme. The enzyme was first discovered in those cell membranes where there are active transport processes: in the brush border of enterocytes and cells of the proximal tubules of the kidneys, in the cilia of the cells of the vas deferens, in the endothelium of arterioles. Alkaline phosphatase is also found in the endoplasmic reticulum, Golgi apparatus, pinocytotic vesicles and enterocyte lysosomes, granules of neutrophilic leukocytes, in the cell membrane and pinocytotic vesicles of smooth muscle cells. The method for determining the activity of alkaline phosphatase consists of the following steps.

Sampling
After sampling (biopsy, material from experimental animals), the tissue is treated with maximum speed, because enzyme activity may change under the influence of autolytic processes. The volume or quantity of material is determined by the objectives of the study, the nature of the preparation of the tissue and the type of organ in the study of intracellular localization of enzymes. The thickness of the tissue pieces should be minimal; no more than 1 cm.

Fixation
Fixation is used in the study of intracellular localization of enzymes (Lojda, 1965). Aldehydes or mixtures thereof are usually used for fixation. Previously used fixation in acetone followed by pouring into paraffin or celloidin is currently not recommended.

 When fixing in formaldehyde, the results (enzymatic activity, preservation of the morphological structure) depend on the concentration, pH, fixation time and temperature.

 Typically, the formaldehyde concentration is 4%. This concentration in the best way meets the requirements for the preservation of the structure and enzymatic activity. In higher concentrations, the fixer suppresses the activity of enzymes too much, and causes artifacts to wrinkle, while in lower concentrations it does not completely immobilize the enzyme and does not ensure proper preservation of the tissue.

 It is best to prepare a solution of formaldehyde 4% in borate buffer with a pH of 9-9.2. Shake it vigorously and filter it.

Fixation is carried out at a temperature of + 4 ^o C (in the refrigerator); at higher temperatures, the enzyme inactivates much faster.

 Fixation time depends on the type of enzyme and organ under study, as well as on the size of the tissue block. For blocks whose thickness does not exceed 5 mm, the fixation time in formaldehyde is 12 hours. Longer fixation causes tissue wrinkling and substantial inactivation of the enzyme. During the first 10-30 minutes, the enzymatic activity decreases in proportion to the increase in fixation time, and then remains relatively stable. Therefore, the fixation time can be increased, because hydrolase inactivation does not occur.

 Then pieces are removed from the fixative, “dried” with filter paper and transferred to a cooled borate buffer (pH = 9–9.2).

Flushing and priming
To increase the residual activity of the enzyme, the tissue block should be thoroughly washed after fixation. Washing increases the residual activity by two or even three times, which makes it possible to detect cells with low enzymatic activity. The washing is carried out in running water or at + 4 ^o C in distilled water, borate buffer (pH = 9-9.2).

 The washing time depends on the fixation time, so when fixing 12 hours it is necessary to rinse the same time.

Before pouring, tissue preparations are dehydrated at +4 ^o С for 30-45 min in 50% acetone, and then in three shifts of 100% acetone for 3-24 hours. In the last change of acetone, the material is left for 1-2 hours at room temperature. The tissue blocks are illuminated in three shifts (10 min each) of benzene, toluene or xylene, soaked in filter paper, placed in paraffin for 30-45 minutes at 56 ^o C and transferred to the vessels for filling with a new change of paraffin.

Paraffin Slices
The paraffin block (on the block) is strengthened in the microtome object holder, the block length is placed parallel or perpendicular to the microtome sled.

 Then strengthen the knife perpendicular to the length of the microtome or obliquely. An oblique position is indicated for cutting dense fabrics. The block and knife are brought together for contact, the surface is leveled by cutting thick sections, after pieces of tissue or organ are found, they are transferred to a section of the required thickness (from 3-5 to 15 microns).

 The obtained sections with a brush or dissecting needle are removed from the razor microtome and in a cup with warm distilled water. For gluing, a slice is caught from water on a previously prepared slide. Sections on the glass are left to fix for 6-24 hours at room temperature.

Buffer Preparation
At the beginning, the pH-150 ionomer was calibrated. The buffer mixture included: sodium tetraborate Na ₂ В ₄ O ₇ • 10 Н ₂ О 0.025 M (with a molecular weight of 382), from which solution A was prepared, 0.953 g was taken for this, and dissolved in distilled water in a volume of 100 ml; then a solution of B was prepared, consisting of 0.1 M Hcl (molecular weight 36.465).

 Then, 50 ml of solution A — sodium tetraborate and solution B 0.1 M Hcl 4.6–5 ml were adjusted to 100 ml with distilled water, pH 9–9.2. It is very important to obtain a pH within these limits to activate the enzyme, alkaline phosphatase.

 Preparation of the incubation mixture.

Solution A. To 25 mg of naphthyl phosphate add 25 ml of buffer - sodium tetraborate with a pH of 9.0 to 9.2. Solution B - 50 mg of blue solid C is dissolved in 25 ml of tetraborate buffer with a pH of 9.0-9.2. Filter in a dark place. Solutions A and B are mixed. Incubate gastroslics from 30 to 45 minutes at a temperature of 20 ^o C after incubation.

Incubation of sections
The incubation medium is drained and the sections rinsed in 3 shifts of distilled water. Then placed in 4% formaldehyde to remove gas bubbles in the slices. Rinse in distilled water. Then the nuclei are additionally stained with solid red or neutral red 1% and the quality reaction to alkaline phosphatase is evaluated by color.

 Results: depending on the diazonium salt and, to some extent, on the influence of the substrate, the tissue structure has enzymatic activity, it is stained blue-violet with a strong blue B.

Claims (1)

A method for detecting alkaline phosphatase in animal immunocompetent organs, which involves the study of histosections by preparing tissue, taking, processing, fixing, washing and filling the sample, incubating it in a medium consisting of buffer solution, distilled water, naphthyl phosphate and neutral strong blue B, washing, additional staining and evaluating the qualitative reaction to alkaline phosphatase by color, characterized in that sodium tetraborate Na ₂ B is used to prepare the buffer solution ₄ O ₇ • 10 H ₂ O, which is taken in the ratio with naphthyl phosphate 1: 1.