Patent RU-2563679-C2 (RU)
Inventor: Lazutkin Aleksandr Alekseevich
IPC: G01N1/30
Filed: 2013-12-11
Granted: 2015-09-20
Abstract: FIELD: chemistry. 
     SUBSTANCE: group of inventions relates to experimental biology and medicine for preparing whole amounts of biological tissues for optical projection tomography, confocal, multi-photonic and plane optical microscopy and can be used for preclinical tests of pharmacological preparations and evaluation of physiological effects on the body, as well as for working with biopsy material for diagnostic and research purposes. A method for immunohistochemical colouring of whole amounts of biological tissue samples includes fixing tissue with paraformaldehyde solution. The method also includes postfixation, which is first carried out with 0.5-1% paraformaldehyde for 48 hours at 4°C, and then after washing three times with a phosphate buffer at room temperature, in a solution of 20% dimethylsulphoxide in 100% methanol for 1-2 hours at room temperature. The tissue sample is then bleached in a solution with the ratio 100% methanol:dimethylsulphoxide:30% H 2 O 2 =4:1:1, for 2-4 hours in bright light at room temperature until complete discolouration. The sample is then washed 3 times for 60 minutes each in 100% methanol at room temperature and frozen for at least 1 hour at -70…-80°C, and then rehydrated successively in 50, 25 and 12.5% methanol solutions in a phosphate buffer for 45-60 minutes each at room temperature. The samples are then washed three times from methanol with phosphate buffer, two times for 1-2 hours each at room temperature, and then the last time for one night at 4°C. Subsequent permeabilisation of the sample is carried out in phosphate buffer containing 2% Triton X-100, 5% normal serum, 5% dimethylsulphoxide, for 2 hours at room temperature. The sample is then washed three times in phosphate buffer containing 0.2% Triton X-100, twice for 30-60 minutes each at room temperature and the last time for one night at 4°C. The sample then undergoes incubation successively in primary and secondary antibody solutions prepared on a buffer which consists of 0.05 M Tris-HCl with pH 7.5; 0.15 M NaCl; 0.2% Triton X-100, 5% dimethylsulphoxide and 2.5-5.0% normal serum of the host of the secondary antibodies, at 4°C for 2-7 days, while washing after each incubation in a buffer consisting of 0.05 M Tris-HCl with pH 7.5; 0.15 M NaCl; 0.2% Triton X-100, first 3 times of 60 minutes each at room temperature, and then for one night at 4°C. The coloured samples are dehydrated successively in 50, 96% ethanol solutions in phosphate buffer for 45-60 minutes each at room temperature. The samples are then held first in 100% ethanol at room temperature 2-3 times for 45-60 minutes each, and then in 2-butoxyethanol at 4°C for 12-18 hours. The dehydrated samples are brightened at room temperature in a mixture of benzyl benzoate and benzyl alcohol, taken in ratio of 2:1, for 3-4 hours. The colouring method, starting from the postfixation procedure, is carried out while mixing at a rate of 650-800 rpm. A second version of the method includes carrying out incubation in a solution of fluorescent labelled primary antibodies, which are prepared on a buffer consisting of 0.05 M Tris-HCl with pH 7.5; 0.15 M NaCl; 0.2% Triton X-100, 5% dimethylsulphoxide and 2.5-5.0% normal serum, at 4°C for 2-7 days. 
     EFFECT: colouring whole amounts of biological tissue samples while maintaining morphology thereof and providing a wider range of detected antigens, enabling quantitative analysis of immunohistochemically detected labels owing to a high signal-to-background ratio and low autofluorescence. 
     18 cl, 2 dwg, 2 ex