METHOD FOR THE COMBINED DETECTION OF KUPFFER CELLS AND CONNECTIVE TISSUE COLLAGEN FIBERS ON HUMAN LIVER HISTOLOGICAL PREPARATIONS Russian patent published in 2025 - IPC G01N33/52 A61B5/00 

FIELD: medical science.

SUBSTANCE: invention concerns a method for the combined detection of Kupffer cells and collagen fibers of connective tissue on histological preparations of the human liver. The preparation is fixed in 10% formalin, dehydrated, poured into paraffin, histological sections are prepared with a thickness of 5 to 10 micrometers, applied to slides with an adhesive coating, dried in a thermostat at 40 ° C, paraffin is removed from the sections, conducted using low-concentration alcohols to distilled water, incubated in 3 in an aqueous solution of hydrogen peroxide for 5 minutes at room temperature. The preparations are washed in distilled water for 5 minutes, the preparations are washed in 0.01 M phosphate-salt buffer with pH = 7.4 for 5 minutes, and a solution of rabbit monoclonal antibodies against the calcium-binding protein Iba1 of clone JM36-62 is applied to the slices at a concentration of 1 mg /l. The preparations are incubated in a wet chamber with 5-10 ml of distilled water added to it in a thermostat at an elevated temperature of 35 °C for 20 hours, the preparations are washed in 0.01 M phosphate-salt buffer with pH = 7.4 for 5 minutes, secondary antibodies against rabbit immunoglobulins are applied to the slices and the preparations are incubated in a wet chamber in keep the thermostat at 27 °C for 40 minutes. The preparations are washed sequentially in 0.01 M phosphate-salt buffer with pH = 7.4 for 5 minutes and in distilled water for 5 minutes, and a working solution of chromogen 3,3'-diaminobenzidine tetrahydrochloride is applied to the slices, monitoring the development of the reaction under a microscope. The preparations are washed in two portions of distilled water, 1% aqueous solution of phosphoric acid is applied to the slices for 15 minutes at room temperature, the solution is removed from the slices and a freshly prepared 1% aqueous solution of aniline blue dye is applied to them with the addition of glacial acetic acid at the rate of 400 µl of acid per 100 ml of dye solution, incubation of the slices in dye solution for 7 minutes at room temperature, rinsing in distilled water for 5 minutes, dehydration in alcohols of rising strength, enlightenment in xylene and confinement in a permanent environment. Preparations are analyzed using light microscopy. At the same time, the immunopositive cells localized in the sinusoids of the liver and colored brown are Kupffer cells, the collagen fibers of the connective tissue are colored a rich blue.

EFFECT: invention provides good reproducibility and high selectivity for detecting Kupffer cells and collagen fibers of connective tissue, suitable for use on human material. The method is simple to perform, affordable, and significantly reduces the cost of the technique and its labor costs. The Kupffer cells and collagen fibers visualized in this way can be analyzed quantitatively.

1 cl, 1 dwg