Stabilization method for biological samples by combination of heating and chemical fixation
The present invention provides methods for stabilizing a biological sample for analysis. The invention more particularly provides methods combining heat treatment and chemical fixation of biological samples in order to maintain protein primary structure and post-translational modifications, such as protein phosphorylations.
1. A method for stabilizing a biological sample for analysis, the method comprising:
a) extracting a biological sample from an organism;
b) heating the sample to a temperature of at least about 70° C. within 3 minutes from the time of extraction of the sample from the organism to arrest enzymatic processes; and
c) submitting the heated sample to chemical fixation.
2. The method according to claim 1 , wherein the method is for stabilizing a biological sample for histological analysis.
3. The method according to claim 1 , wherein the method further comprises analizing the fixed sample a histochemical assay and/or an immunohistochemical assay.
4. The method according to claim 1 , wherein the biological sample comprises at least one protein having a post-translational modification.
5. The method according to claim 4 , wherein the post-translational modification comprises phosphorylation.
6. The method according to claim 1 , wherein the chemical fixation is performed using a cross-linking agent.
7. The method according to claim 6 , wherein the cross-linking agent is an aldehyde.
8. The method according to claim 7 , wherein the aldehyde is formaldehyde or glutaraldehyde.
9. The method of claim 1 , wherein the sample is heated to a temperature of at least 70° C. within 2 minutes from the time of extraction of the sample from the organism.
10. The method of claim 1 , wherein the sample is heated to a temperature of at least 70° C. within 2 minutes from initiation of heating.
11. The method of claim 1 , wherein heating arrests at least 60% of enzymatic activity of the sample.
12. The method of claim 1 , wherein heating arrests at least 70% of enzymatic activity of the sample.
13. The method of claim 1 , wherein
the sample comprises a plurality of biological macromolecules, the biological molecules comprising a primary structure; and
heating degrades the primary structure of less than 5% of the plurality of biological macromolecules.
14. The method of claim 13 , wherein heating degrades the primary structure of less than 2% of the plurality of biological macromolecules.
15. The method of claim 1 , wherein heating comprises conduction, convection, or radiation.
16. The method of claim 1 , wherein
heating comprises conduction; and
the biological sample comprises a shape such that no part of the sample is more than 1 cm from the source of conductive heat.
17. The method of claim 1 , wherein heating comprises conduction;
and the biological sample comprises a shape such that no part of the sample is more than 9 mm from the source of conductive heat.
18. The method of claim 1 , wherein the sample is heated to a temperature of about 70° C. up to about 100° C.
19. The method of claim 1 , wherein the sample is heated to a temperature of at least about 80° C.
20. The method of claim 1 , further comprising:
placing the extracted sample in a container prior to heating; and
evacuating air from the container such that the sample directly contacts the container.
21. The method of claim 1 , wherein the sample comprises a proteolytic enzyme.
22. The method of claim 1 , wherein the sample comprises a tissue selected from the group consisting of tissue, muscle, bone, bone marrow, tooth, hair, skin, brain, kidney, liver, stomach, intestine, reproductive organ, and pancreas.
23. The method of claim 1 , wherein the sample comprises a fluid selected from the group consisting of tears, saliva, blood, semen, sweat, and urine.
24. The method of claim 1 , wherein the organism is selected from the group consisting of mammal, reptile, invertebrate, fish, insect, and bird.
25. The method of claim 1 , wherein the organism is selected from the group consisting of human, primate, rabbit, sheep, dog, cat, horse, monkey, mouse, and rat.