METHOD OF IMMUNOSTAINING A BIOLOGICAL MATERIAL FOR CONFOCAL MICROSCOPY Russian patent published in 2019 - IPC G01N33/53 

FIELD: biology.

SUBSTANCE: invention relates to an improved method of immunostaining a biological material for use in confocal microscopy. Method involves preparation of material, fixation, removal of retainer, blocking of non-specific binding of antibodies, incubation with primary antibodies in blocking buffer, washing from primary antibodies, incubation with secondary antibodies in the blocking buffer, washing from secondary antibodies, dehydration, clarification and visualization of the object, according to the invention. Blocking buffer used is a phosphate buffer containing 0.1–1.0 % non-ionic detergent, 20.0 % dimethylsulphoxide, 1.0 % bovine serum albumin, not more than 10.0 % sheep serum and 0.03 % sodium azide. Non-ionic detergents used are Tween-20 or Triton X-100. Fixation is carried out in solution of 4 % paraformaldehyde with methanol on phosphate buffer for 5–6 hours at +4 °C; removal of retainer is performed washing buffer 3 times for 20 minutes at room temperature; non-specific binding of antibodies is blocked for 12 hours with a blocking buffer at +4 °C; incubation with primary antibodies in blocking buffer is carried out for 5–7 days at room temperature and constant stirring, subsequent washing from primary antibodies is carried out in washing buffer, 6 times for 20 minutes, at room temperature; incubation with secondary antibodies in blocking buffer is carried out for 2 days at room temperature and constant stirring, washing from secondary antibodies is 6 times for 20 minutes at room temperature in washing buffer. Removal of retainer, washing from primary and secondary antibodies, as a rule, washing buffer containing phosphate buffer with addition of non-ionic detergents, preserving tissue structure, in concentrations from 0.1 to 1.0 %. Dehydration is carried out by incubating material in 50 % methanol for 10 minutes, and then transferred to 100 % methanol for 20 minutes, followed by replacement of solution with 100 % fresh methanol and held for at least 20 minutes with constant stirring; optical clarification by chemical decolouration is carried out by incubating material in a mixture of benzyl alcohol and benzyl benzoate in ratio of 1:2, for 1 hour at room temperature, with further replacement with fresh solution of benzyl alcohol and benzyl benzoate in ratio of 1:2 and incubated in it during night with constant stirring, after which the object is visualized. Object visualization is usually carried out using confocal microscopy in a combination of modes Z-stack and TILE-scan. Composition of the blocking buffer under the proposed exposure conditions enables to eliminate nonspecific staining throughout the sample, marking only certain structures and obtaining a clear image thereof.

EFFECT: method makes it possible to easily visualize fabric structures lying at a depth of more than 150 mcm, which is an advantage of a method which enables to adapt it for different biological objects and considerably expand range of analyzed biological materials up to 6 cm³ in size.

5 cl, 5 dwg, 1 tbl, 5 ex