Immunostaining-DAKO Envision Plus Kits
Notes:
1. For each tissue array, 300-400ul of each incubating solution is needed.
2. Procedure is done at room temperature except for microwaving steps.
3. EnVision + kit catalog numbers: Rabbit K4010 (150tests) or K4011 (1100 tests) and
Mouse K4006 (150 tests) or K4007 (1100 tests)
Staining procedure
1. Deparaffinize slides through three changes of xylene, incubating slides10 min in each change.
2. Hydrate slides to water by dipping them twenty times in each of two changes of 100% ethanol,
two changes of 95% ethanol, one of 80% ethanol, one of 70 % ethanol, and two changes of
distilled water.
3. Place slides in microwave slide dish (plastic works best with a plastic slide rack) and cover with
citrate buffer.
4. Microwave on high power for 3 minutes if one rack of slides, 8 minutes if two racks of slides and
13 minutes if three racks of slides.
5. Continue microwaving at 80% power for 12 minutes. (Times may vary by microwave, but the
important thing is to make sure the slides boil rapidly for 10 minutes minimum and do not dry
out. An additional container of citrate buffer may be microwaved together with the slides to
allow for refilling of the slide dishes.)
6. Cool for a minimum of 30 minutes. Cooling time is critical.
7. Rinse in distilled water, X2.
8. Place in PBS.
9. Dry excess PBS from slides and circle tissue with hydrophobic pen leaving 1mm space between
tissue and circle. Be sure that tissue never dries out.
10. Block endogenous peroxidase activity by covering circled area with solution from bottle #1 of
DAKO kit and incubating for 5 minutes.
11. Rinse in PBS, X2
12. Cover circled area with primary antibody or negative control solution and incubate for 30
minutes.
13. Rinse in PBS, X2
14. Cover circled area with bottle #2 from DAKO kit and incubate for 30 minutes.
15. Rinse in PBS, X2
16. Prepare DAB according to number of slides using bottles #3a and #3b from DAKO kit. (1ml of
bottle #3a to 1 drop of bottle 3b)
17. Cover circled area with prepared DAB and incubate 10 minutes. Keeping this time constant
allows for more accurate duplication of titers.
18. Rinse with distilled water, X2
19. Counterstain with hematoxylin (DAKO S3309) for 10 seconds.
20. Rinse with tap water, X3.
21. Dip slides five times in dilute ammonia water or lithium carbonate water (a slightly basic
solution is all that is needed) to blue.
22. Rinse in tap water, X3.
23. Dehydrate slides by dipping them twenty times in three changes of 95% ethanol followed by
three changes of 100% ethanol.
24. Clear slides by dipping twenty times in three changes of xylene.
25. Coverslip slides with Protex or other xylene-compatible mounting medium.
Solutions:
Citrate Buffer = 10um citrate buffer pH6.0
Method:
Measure 1 liter distilled water
Add 2.1gm of citrate acid monohydrate (mw=210.1)
Adjust pH to 6.0 with sodium hydroxide or HCl as necessary
Antibody dilutant = 1% albumin in PBS with 0.1% NaAzide
Method
Make stock 10% NaAzide
Measure 10ml distilled water
Add 1gm powdered NaAzide
Measure 100ml PBS working solution
Add 1gm powered albumin
Add 1 ml 10% stock NaAzide
25X PBS (phosphate buffer)
360g Sodium Chloride
66g Sodium phosphate (monobasic, monohydrate)
376g Potassium phosphate (dibasic)
add distilled water up to 2 liters. (pH should be around 7.3)
1X PBS (working solution)
dilute the 25X PBS 1:25 with H2O