John M. Sanders B.S. Cardiovascular Research Center, University of Virginia Charlottesville, VA.
The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies.
1. Deparaffinize and rehydrate 5 min tray
2 x xylene
2 x absolute ETOH
2 x 95 % ETOH
2 x 70 % ETOH
2 x milliQ H2O
1. Quench endogenous peroxidase activity
200 ml methanol + 3 ml (30% H2O2) in slide tub
- make just before use, Incubate 30 minutes, RT
2a. wash H2O 5 min room temp.
1. Make up antigen unmasking solution (Vector Labs; 4oC) OR other antigen retrieval techniques
- Shake well before measuring
- 320 milliQ H2O + 3 ml unmasking solution
- Mix by inversion
1. Rinse in milliQ H2O in tub
2. In slide tub with “fill” mark, fill all 24 slots in slide holder with “blank slides” if necessary, add unmasking solution to fill line, COVER and microwave for 20 minutes Replenish with dH20 at:
- 15 minutes remaining
1. Cool slides in tub, covered, at room temp for 1 hour
2. During 1 hour, make up 0.5% FSGO (fish skin gelatin oil) in PBS (1 500 ml bottle PBS + 2.5 ml FSGO; allow 15 min for FSGO to come out of pipet)
3. Transfer to tub with plain room temperature PBS, 5 min
4. Make up avidin blocking solution:
For 1 ml:
- 1 ml PBS/FSGO
- 100 ul normal serum of same species as secondary antibody
4 drops avidin blocking solution (Vector Labs)
- Invert tube to mix
5. Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST
6. Add blocking solution within circles; 1 hour in humidified chamber at room temperature
7. Aspirate off blocking solution
8. Add primary antibody to vessels on slides.
For 4 slides: antibody
- PBS/FSGO 1 ml
- normal serum 100 ul
- biotin blocking solution (Vector Labs) 4 drops
9. Incubate 4 oC overnight in humidified chamber.
1. Aspirate primary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate 2. Make up secondary antibody:
- 5 ul secondary antibody
- 100 ul normal serum
- 1 ml FSGO/PBS
3. Blot slides with kimwipe and apply secondary antibody; incubate in humidified chamber 1 hour, room temperature
4. During incubation, make up ABC solution from kit: per ml:
- 1 ml plain PBS
- 20 ul A solution
- 20 ul B solution
Allow to rock at room temperature for at least 30 min in order to form complex
5. Aspirate secondary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate
6. Blot slides and apply ABC; incubate 30 min at room temperature
7. Make up DAB solution: Approximately 30 min before using; add H2 O2 just before use
- 10 ml plain PBS
- 1 DAB tablet
- 7.5 ul H2 O2 (30%)
Test DAB and ABC by adding small amount of DAB to a small amount of left over ABC, should turn dark brown immediately.
8. Wash with agitation in plain PBS, 5 min room temperature
9. Blot slides and apply DAB solution, incubate 5 min room temperature
10. Wash in dH20, 5 min room temperature
11. Counterstain in filtered (#1 Whatman) Hematoxylin I (not Mayer’s); 4-5 min
12. Rinse in running tap water until clear, 4-5 min. Return hematoxylin to bottle for reuse
13. Blueing if needed for 1 min; wash in tap water
14. Dehydrate, 5 minutes each:
- 3 x absolute ETOH
- 2 x xylene
Barringhaus K, Phillips JW, Thatte J, Sanders J, Czarnik A, Bennett D. Ley K, Sarembock I. a4b1 Integrin (VLA-4) Blockade Attenuates both Early and Late Leukocyte Recruitment and Neotimal Growth following Carotid Injury in Apoliprotein E (-/-) Mice. Journal of Vascular Research 2004 41:252-260.
Manka D, Forlow SB, Sanders J, Hurwitz D, Bennett D, Green S, Ley K. Sarembock I. Role of Platelet P-Selectin in the Response to Arterial Injury in Apoliprotein-E Deficient Mice. Arteriosclerosis, Thrombosis, and Vascular Biology 2004 24:1124.