Methods for examining podocyte foot processes in human renal samples using conventional optical microscopy
The invention provides a method for preparing an expanded renal (kidney) tissue sample suitable for microscopic analysis. Expanding the kidney sample can be achieved by binding, e.g., anchoring, key biomolecules to a polymer network and swelling, or expanding, the polymer network, thereby moving the biomolecules apart as further described herein. As the biomolecules are anchored to the polymer network, isotropic expansion of the polymer network retains the spatial orientation of the biomolecules resulting in an expanded, or enlarged, kidney sample.
1. A method for measuring tertiary foot process effacement using conventional microscopy, the method comprising the steps of
(a) staining a kidney tissue sample with foot process-specific antibodies;
(b) contacting the stained kidney tissue sample with a bi-functional linker;
(c) permeating the contacted kidney tissue sample with a solution comprising precursors of a swellable material,
(d) polymerizing the precursors to form a swellable polymer within the permeated kidney tissue sample, wherein the bi-functional linker attaches or crosslinks to the swellable material;
(e) incubating the permeated kidney tissue sample in (d) with a non-specific protease in a buffer comprising 5 mM to about 100 mM metal ion chelator, a nonionic surfactant, and a monovalent salt under conditions that allow the non-specific protease to digest and homogenize the kidney tissue sample-polymer complex;
(f) expanding the incubated kidney tissue sample by contacting the swellable polymer with a solvent or liquid to cause the swellable polymer to swell;
(g) subjecting the expanded kidney tissue sample to microscopic analysis; and
(h) measuring tertiary foot process effacement in the expanded kidney tissue sample.
2. The method according to claim 1 , wherein the microscopic analysis comprises one or more of analysis of general morphology, measuring widths of individual of tertiary foot processes, and measuring intervals between adjacent foot processes.
3. The method according to claim 2 , wherein the analysis detects one or more podocyte disease or disorders selected from the group consisting of loss of podocytes or podocytopenia, podocyte mutation, an increase in foot process width, a decrease in slit diaphragm length, effacement or a diminution of podocyte density, and decrease in a podocyte number.
4. The method according to claim 1 , wherein the bi-functional linker attaches to the swellable material during polymerization.
5. The method according to claim 1 , wherein the bi-functional linker attaches to the swellable material after polymerization.
6. The method according to claim 1 , wherein the bi-functional linker comprises a protein-reactive chemical moiety and a gel-reactive chemical moiety.
7. The method according to claim 1 , wherein the protein-reactive chemical moiety is a succinimidyl ester of 6-((acryloyl)amino) hexanoic acid (AcX).
8. The method according to claim 1 , wherein the kidney tissue sample is a previously preserved clinical sample.
9. The method according to claim 8 , wherein the kidney tissue sample is a formalin fixed paraffin embedded (FFPE) or a hematoxylin and eosin (H&E) stained tissue sample, or a fresh frozen sample.
10. The method according to claim 9 , wherein prior to the measuring the kidney tissue sample is subjected to (i) de-coverslipping the kidney tissue sample if it is mounted; (ii) treating the kidney tissue sample to mounting medium removal; (iii) treating the kidney tissue sample to re-hydration if step (ii) is performed; and (iv) subjecting the kidney tissue sample to antigen-retrieval.
11. The method according to claim 1 , wherein the kidney tissue sample is incubated with about 1 to about 100 U/ml of a non-specific protease in a buffer having a pH between about 4 and about 12, the buffer comprising about 5 mM to about 100 mM of a metal ion chelator; about 0.1% to about 1.0% of a nonionic surfactant; and about 0.05 M to about 1.0 M monovalent salt.
12. The method according to claim 1 , wherein the solution permeating the kidney tissue sample further comprises an inhibitor, an initiator, and an accelerator.
13. The method according to claim 12 , wherein the inhibitor comprises 4-hydroxy-2,2,6,6,-tetramethylpiperidin-1-oxyl.
14. The method according to claim 12 , wherein the initiator comprises ammonium persulfate.
15. The method according to claim 12 , wherein the accelerator comprises tetramethylethylenediamine (TEMED).