Immunofluorescence assays
Provided herein are immunofluorescence assays that do not use an intensely colored lipophilic dye, and that result in a significant reduction of fluorescence due to aldehyde fixation, collagen, elastin, and/or red blood cells. In certain embodiments of the present invention, a two-step treatment of the tissue is contemplated that leads to the incorporation of reduced heteropoly acids or salts thereof (e.g., phosphomolybdate species) in the tissue. This reagent binds tightly to hydrophilic regions of the tissue and effectively quenches the fluorescence in those regions. In doing so, the overall signal-to-noise ratio for the immunofluorescence assay is increased.
1. A method for lowering/reducing/masking/suppressing tissue autofluorescence in an immunofluorescence assay, said method comprising, before or during conducting said immunofluorescence assay:
providing a tissue comprising lung, liver, heart, nervous system, skin, lymph gland, musculoskeletal, spleen, eye, sinus, nasal mucosa, larynx, gastrointestinal tract tissue, reproductive organ, breast, prostate, salivary gland, tonsil, or kidney tissue;
applying a heteropoly acid or salt thereof to said tissue, wherein the heteropoly acid comprises phosphomolybdic acid, phosphotungstic acid, silicomolybdic acid, silicotungstic acid, or a mixture of any two or more thereof or salts thereof;
reducing at least a portion of said heteropoly acid or salt thereof to a reduced form with a reducing agent; and
measuring the immunofluorescence of the tissue.
2. The method of claim 1 wherein the reducing agent comprises an ascorbic acid or salts thereof.
3. The method of claim 1 wherein said heteropoly acid or salt thereof is applied to said tissue before measuring the fluorescence of the tissue.
4. The method of claim 3 wherein said heteropoly acid or salt is applied to said tissue before the reducing step.
5. The method of claim 3 wherein said heteropoly acid or salt thereof is applied to said tissue in reduced form.
6. The method of claim 1 wherein said heteropoly acid or salt thereof is applied to said tissue while conducting said immunofluorescence assay.
7. The method of claim 6 wherein said heteropoly acid or salt thereof is subsequently reduced.
8. The method of claim 6 wherein said heteropoly acid or salt thereof is applied in reduced form.
9. The method of claim 1 wherein when the heteropoly acid is applied in salt form, the salt comprises a counter-ion for said heteropoly anion which is a monovalent metal ion; a divalent metal ion; a trivalent metal ion; a quaternary ammonium cation; a quaternary phosphonium cation; or a mixture of any two or more thereof.
10. The method of claim 1 wherein said heteropoly acid or salt thereof is applied to said tissue as a solution.
11. The method according to claim 10 wherein said heteropoly acid or heteropoly anion is applied as a solution in a range of concentrations from 0.002% (w/v) up to a saturated solution.
12. The method of claim 1 wherein said heteropoly acid or salt thereof is applied to said tissue as a suspension.
13. The method of claim 1 wherein said heteropoly acid or salt thereof is applied to said tissue as a solid.
14. The method of claim 1 wherein said heteropoly acid or salt thereof binds to hydrophilic regions of the tissue.