METHOD FOR PREPARING LARGE SAMPLES OF REGENERATING BONE FOR SCANNING ELECTRON MICROSCOPY Russian patent published in 2021 - IPC G01N1/28 

FIELD: biology; medicine.

SUBSTANCE: invention relates to the field of biology and medicine, more specifically to the field of traumatology, orthopedics and dentistry, as well as regenerative medicine, and can be used when conducting scanning electron microscopy to study bone reparative regeneration. Disclosed is a method for preparing large samples of regenerating skeletal connective tissue, bone and surrounding muscle and / or nervous tissue and organs for studying the morphology of biological structures in the area of regeneration on the slice plane passing through the entire sample using the method of scanning electron microscopy, including at least the following steps: fixation of the sample; rinsing the sample; postfix; demineralization of the sample in a mixture of aqueous solutions of 8% hydrochloric acid and 8% oxalic acid in a 1:1 ratio for 24-72 hours; soaking the sample with a 1.6M sucrose solution for 1 hour; programmed freezing of the sample at a rate of 1°C per minute; cryotome sample processing; dehydration in 70-80% ethyl alcohol at a temperature of -25°C for at least 24 hours; washing sucrose - keeping the sample for 4-6 hours in 70% ethanol solution at room temperature; holding the sample in alcohol with a concentration of 85% for 4 hours, in ethyl alcohol with a concentration of 96% for 2 hours, a mixture of absolute isopropyl alcohol - diethyl ether for 20 minutes, diethyl ether for 10 minutes, a mixture of diethyl ether-camphene in for 30 minutes, then impregnation with molten camphene is carried out at 42°-45°C for 8-48 hours; drying with sublimation of camphene under controlled conditions in an upward flow of dry and free of contaminant particles inside the desiccator with a 2-fold change of the air mixture per hour at a temperature of 30°C for at least 48 hours.

EFFECT: development of a method for preparing large samples of regenerating skeletal connective tissue for studying the morphology of the bone surface using the method of scanning electron microscopy, which makes it possible to prepare a sample with minimal deformation of histological structures and possibly complete preservation of their true architectonics, namely, with the preservation of the natural interposition of cellular and matrix components in these biological objects, as well as elimination of gross deformations of samples and artifacts of the tissue complex in preparation for scanning electron microscopic examination.

11 cl, 2 dwg, 1 ex