Methods for phenotyping of intact whole tissues
In various embodiments, the present application teaches methods and compositions for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically-transparent, thereby exposing their cellular structure with intact connectivity. In some embodiments, the present application teaches PACT, a protocol for passive tissue clearing and immunostaining of intact organs. In other embodiments, the present application teaches RIMS, a refractive index matching media for imaging thick tissue. In yet other embodiments, the application teaches PARS, a method for whole-body clearing and immunolabeling.
1. A method for modifying the structural and/or optical characteristics of a tissue, comprising:
applying a fixing solution comprising paraformaldehyde (PFA) to the tissue, thereby forming fixed tissue;
applying a hydrogel monomer solution comprising 4% acrylamide to the fixed tissue, thereby forming hydrogel treated tissue;
initiating polymerization of hydrogel monomers;
applying a detergent comprising 8% sodium dodecyl sulfate (SDS) to the hydrogel treated tissue; and
passively clearing the tissue without the use of electrophoresis, thereby forming cleared tissue.
2. The method of claim 1 , further comprising applying a photoinitiator solution comprising 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride to the fixed tissue.
3. The method of claim 1 , further comprising
placing the hydrogel treated tissue into a substantially air tight chamber, and
introducing nitrogen into the substantially air tight chamber, thereby forming a de-gassed tissue.
4. The method of claim 3 , further comprising incubating the de-gassed tissue at from 15° C. to 60° C., thereby forming incubated tissue.
5. The method of claim 4 , further comprising washing the incubated tissue with PBS, thereby forming washed and incubated tissue.
6. The method of claim 1 , further comprising applying PBS to the cleared tissue, thereby forming cleared and washed tissue.
7. The method of claim 6 , further comprising applying imaging media to the cleared and washed tissue, wherein the imaging media comprises: (1) 1-N,3-N-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetamido]-2,4,6-triiodobenzene-1,3-dicarboxamide or 5-(N-2,3-Dihydroxypropylacetamido)-2,4,6-triiodo-N,N′-bis(2,3-dihydroxypropyl)isophthalamide, (2) phosphate buffer; (3) tween-20; (4) sodium azide; and optionally (5) sodium hydroxide.
8. The method of claim 7 , wherein the concentration of 1-N,3-N-bis(2,3-dihydroxypropyl)-5-[N-(2,3-dihydroxypropyl)acetamido]-2,4,6-triiodobenzene-1,3-dicarboxamide or 5-(N-2,3-Dihydroxypropylacetamido)-2,4,6-triiodo-N,N′-bis(2,3-dihydroxypropyl)isophthalamide is from 10 to 100% w/v.
9. The method of claim 8 , wherein the tissue is obtained from a biopsy.
10. The method of claim 9 , wherein the tissue is cancerous or precancerous.
11. A method for immunostaining tissue, comprising applying a solution comprising a primary antibody to the cleared and washed tissue of claim 6 , thereby forming an antibody-bound tissue.
12. The method of claim 11 , further comprising rinsing the antibody-bound tissue with a buffer solution.
13. The method of claim 12 , further comprising applying a solution comprising a secondary antibody to the antibody-bound tissue that has been washed with said buffer solution, wherein the secondary antibody is labeled with a visualizable marker.
14. The method of claim 13 , wherein the visualizable marker is fluorescent.
15. The method of claim 11 , wherein the primary antibody is labeled with a visualizable marker.
16. The method of claim 15 , wherein the visualizable marker is fluorescent.
17. The method of any one of claims 11 - 15 , wherein the tissue is obtained from a biopsy.
18. A method for visualizing immunostained tissue, comprising:
utilizing a microscope to visualize immunostained tissue prepared according to the method of claim 13 .
19. The method of claim 18 , wherein the microscope is utilized to implement a form of microscopy selected from the group consisting of confocal microscopy, spinning disk microscopy, epi-fluorescence microscopy, light field microscopy, light-sheet microscopy, multiphoton microscopy.