Automated method for detecting apoptosis in cells

• US 7,687,243 B1
• Filed: 06/06/2006
• Issued: 03/30/2010
• Est. Priority Date: 06/06/2005
• Status: Active Grant

• Current Assignee: Tonia M. Crook
• Original Assignee: Tonia M. Crook
• Inventors: Crook, Tonia M.
• Primary Examiner(s): Lam; Ann Y
• Application Number:
  US11/447,556
• Time in Patent Office: 1,393 Days
• Field of Search: 435/7.9, 435/4, 435/7.1, 435/7.2, 435/283.1, 435/289.1, 435/287.3
• US Class Current: 435/7.9
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism

Abstract

A TUNEL assay method is claimed and described. The method comprises loading slides having mounted tissue samples into a primary stainer and testing tissue samples for apoptosis using a stainer. In one embodiment, the method includes using a stainer having a plurality of reservoirs. A reagent reservoir is loaded with anti-conjugate antibody reporter. A slide tray of the stainer is loaded with a slide having a tissue sample. The stainer adds anti-conjugate antibody reporter to the sample for a duration.

Claims

1. A TUNEL assay method comprising:

adjusting the relative humidity of the room in which said assay is to be performed to about 80%;

adjusting the temperature of the room in which said assay is to be performed to about 22°

C. to about 26°

C.;

loading slides having mounted tissue samples into an immunochemical stainer; and

testing said tissue samples for apoptosis using at least said immunochemical stainer or another histological stainer wherein said testing comprises

adding conjugate-dNTPs to said tissue with said immunochemical stainer or said histological stainer,

adding TdT to said tissue with said immunochemical stainer or said histological stainer;

rinsing said tissue with TBS-tween; and

adding anti-conjugate antibody reporter with said immunochemical stainer or said histological stainer.

2. The assay method of claim 1, wherein said temperature adjusting to about 24°

C.

3. The method of claim 1, wherein said conjugate-dNTPs are digoxigenin-dNTP conjugates.

4. The method of claim 3, wherein said digoxigenin-dNTP conjugates are added to said tissue as a biologically effective amount.

5. The method of claim 1, wherein said TdT is added to said tissue as a biologically effective amount.

6. The method of claim 1, wherein said conjugate-dNTPs and said TdT are added to said tissue as a mix having a volume between about 100 μ

l and about 600 μ

l.

7. The method of claim 1, wherein said anti-conjugate antibody reporter is an anti-digoxigenin antibody reporter added to said tissue in a biologically effective amount.

8. The method of claim 7, wherein said anti-digoxigenin antibody reporter is added as a solution having a volume between about 100 μ

l and about 600 μ

l.

9. The method of claim 1, wherein said anti-conjugate antibody reporter includes a reporter selected from the group consisting of fluorescent, radioactive and colorimetric reporters.

10. The method of claim 1, wherein said anti-conjugate antibody reporter includes a peroxidase reporter capable of reporting in the presence of a chromogenic substrate.

11. The method of claim 1, wherein said anti-conjugate antibody reporter is added for a duration of between about 25 and about 50 minutes.

12. The method of claim 11, wherein said anti-conjugate antibody reporter duration is between about 25 and 35 minutes for rat samples.

13. The method of claim 12, wherein said anti-conjugate antibody reporter duration is about 30 minutes for rat samples.

14. The method of claim 11, wherein said anti-conjugate antibody reporter duration is between about 40 and 50 minutes for mice, primate, and human samples.

15. The method of claim 14, wherein said anti-conjugate antibody reporter duration is about 45 minutes for mice, primate, and human samples.

16. The method of claim 1, further including evaluating for the presence of reporting.

17. The method of claim 16, wherein said evaluating for the presence of reporting includes evaluating for peroxidase-generated stain.

18. The method of claim 1, further including adding proteinase prior to said testing.

19. The method of claim 18, wherein said adding proteinase includes adding between about 100 μ

l and about 600 μ

l of Proteinase K to said tissue for a duration.

20. The method of claim 19, wherein said Proteinase K duration is between about 20 minutes and about 70 minutes.

21. The method of claim 20, wherein said Proteinase K duration is between about 20 minutes and about 40 minutes for mice, primate, and human samples.

22. The method of claim 21, wherein said Proteinase K duration is about 30 minutes for mice, primate, and human samples.

23. The method of claim 20, wherein said Proteinase K duration is between about 50 and about 70 minutes for rat samples.

24. The method of claim 23, wherein said Proteinase K duration is about 60 minutes for rat samples.

25. The method of claim 1, further including deparaffinizing said slides prior to said testing with a stainer.

26. The method of claim 25, wherein said deparaffinizing is achieved automatically by said primary stainer.

27. The method of claim 1, wherein said testing further includes adding a reporter substrate.

28. A TUNEL assay method using a stainer having a plurality of reservoirs, said assay comprising:

loading a reagent reservoir of a primary stainer with anti-conjugate antibody reporter;

loading a slide tray of said primary stainer with a slide having a tissue sample; and

adding said anti-conjugate antibody reporter to said sample for a duration with said primary stainer and further including loading a TdT and conjugate-dNTP mix into another of said reservoirs, and adding said TdT and conjugate mix to said tissue with said stainer prior to the addition of said anti-conjugate antibody reporter for a duration; and

rinsing said tissue with TBS-tween prior to the addition of said anti-conjugate antibody report for a duration.

29. The method of claim 28, wherein said anti-conjugate antibody reporter is an anti-digoxigenin antibody reporter.

30. The method of claim 28, wherein said anti-conjugate antibody reporter is loaded into said reagent reservoir in an amount to provide about 100 μ

l to about 600 μ

l of biologically effective anti-conjugate antibody reporter per sample.

31. The method of claim 30, wherein said anti-conjugate antibody reporter is loaded to provide about 300 μ

l of solution per sample.

32. The method of claim 28, wherein said anti-conjugate antibody reporter duration is between about 20 minutes and about 50 minutes.

33. The method of claim 32, wherein said anti-conjugate antibody reporter duration is in between about 40 minutes and about 50 minutes for mice, primate, and human samples.

34. The method of claim 33, wherein said anti-conjugate antibody reporter duration is about 45 minutes for mice, primate, and human samples.

35. The method of claim 32, wherein said anti-conjugate antibody reporter duration is between about 20 minutes and about 40 minutes for rat samples.

36. The method of claim 35, wherein said anti-conjugate antibody reporter duration is about 30 minutes for rat samples.

37. The method of claim 28, wherein said conjugate-dNTP includes a digoxigenin-dNTP.

38. The method of claim 37, wherein said digoxigenin-dNTP includes a digoxigenin-dUTP.

39. The method of claim 28, wherein said mix is loaded to provide a biologically effective amount of TdT to said tissue.

40. The method of claim 39, wherein said mix amount is administered in a volume between about 100 μ

l to about 600 μ

l per sample.

41. The method of claim 28, wherein said TdT and conjugate dNTP mix duration is about 60 minutes.

42. The method of claim 28, further including loading a proteinase into another of said reservoirs, and

adding said proteinase to said tissue with said stainer prior to the addition of said anti-conjugate antibody reporter for a duration.

43. The method of claim 42, wherein said proteinase includes Proteinase K.

44. The method of claim 43, wherein said Proteinase K is loaded to provide about 100 μ

l to about 600 μ

l per sample.

45. The method of claim 42, wherein said proteinase duration is from about 20 minutes to about 70 minutes.

46. The method of claim 45, wherein said proteinase duration is from about 20 minutes to about 40 minutes for mouse, primate, and human samples.

47. The method of claim 46, wherein said proteinase duration is about 30 minutes for mouse, primate and human samples.

48. The method of claim 47, wherein said proteinase duration is from about 50 minutes to about 70 minutes for rat samples.

49. The method of claim 48, wherein said proteinase duration is about 60 minutes for rat samples.

50. The method of claim 28, further including adding a reporter substrate after the addition of said anti-conjugate antibody reporter.

51. A TUNEL assay method comprising:

adjusting the relative humidity of the room in which said assay is to be performed to about 80%;

adjusting the temperature of the room in which said assay is to be performed to about 22°

C. to about 26°

C.;

loading an immunochemical stainer with reagents for applying to tissue samples, said reagents including buffer, Proteinase K, conjugate-dNTPs, TdT, and, anti-conjugate antibody reporter;

deparaffinizing said samples;

adding Proteinase K to said tissue sample for a duration with said immunochemical stainer;

adding conjugate-dNTPs to said tissue sample for a duration with said immunochemical stainer;

adding TdT to said tissue sample for a duration with said immunochemical stainer;

rinsing said tissue with TBS-tween; and

adding anti-conjugate antibody reporter to said tissue sample for a duration with said immunochemical stainer.