Method Immunohistochemical staining total preparations of a sample of biological tissue (OPTIONS)

摘要

1. A method of immunohistochemical staining of total preparations of biological tissue samples including tissue fixation paraformaldehyde solution postfiksatsiyu, in methanol dehydration, rehydration solutions in methanol, permeabilization, washing with PBS, incubation in the primary antibody solution, washing buffer, incubating the solution of fluorescent secondary antibodies , washing in a buffer dehydration bleaching mixture in benzyl benzoate: benzyl alcohol, in the ratio 2: 1, wherein the first conduct postfiksatsiyu 0.5-1% paraformaldehyde for 24-48 hours at 4 ° C, and then, after washing three times with PBS at room temperature in a solution of 20% dimethyl sulfoxide in 100% methanol for 1-2 hours at 4-20 ° C, then bleached tissue sample in a solution containing 100% - ny methanol: DMSO 30% HOv ratio of 4: 1: 1 for 2-4 hours in the bright light at room temperature until complete discoloration, then washed 3 times for 60 min in 100% methanol at room temperature and do not freeze less than 1 hour at -70 ... -80 ° C, and then rehydrated in a series of 50, 12.5 and 25% solutions of methanol in the phosphate buffer at 45-60 minutes each at room temperature and then samples were washed three times methanol-phosphate buffer and washed twice for 2.1 h at room temperature, and the last wash carried out overnight at 4 ° C, conduct subsequent permeabilization sample in phosphate buffer containing 2% Triton X-100, 5% -ing normal serum, 5% dimethyl sulphoxide, for 2 hours at room temperature, then the sample is washed three times in phosphate buffet