Griffin:Immunohistochemistry Paraffin: Difference between revisions
(New page: '''Immunohistochemistry Protocol''' John M. Sanders B.S. Cardiovascular Research Center, University of Virginia Charlottesville, VA. The procedure below has been proven to be effective ...) |
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'''Immunohistochemistry Protocol''' | '''Immunohistochemistry Protocol''' | ||
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. | The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies. | ||
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200 ml methanol + 3 ml (30% H2O2) in slide tub | 200 ml methanol + 3 ml (30% H2O2) in slide tub | ||
*make just before use | *make just before use, Incubate 30 minutes, RT | ||
Incubate 30 minutes, RT | |||
'''2a.''' wash H2 O 5 min room temp. | '''2a.''' wash H2 O 5 min room temp. | ||
| Line 27: | Line 27: | ||
'''1.''' Make up unmasking solution (Vector Labs; 4oC) | '''1.''' Make up unmasking solution (Vector Labs; 4oC) | ||
Shake well before measuring | *Shake well before measuring | ||
320 milliQ H2O + 3 ml unmasking solution | *320 milliQ H2O + 3 ml unmasking solution | ||
Mix by inversion | *Mix by inversion | ||
'''1.''' Rinse in milliQ H2O in tub | '''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: | '''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 | ||
15 minutes remaining | dH20 at: | ||
11 | *15 minutes remaining | ||
7 | *11 | ||
3 | *7 | ||
1 | *3 | ||
*1 | |||
'''1.''' Cool slides in tub, covered, at room temp for 1 hour | '''1.''' Cool slides in tub, covered, at room temp for 1 hour | ||
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For 1 ml: | For 1 ml: | ||
1 ml PBS/FSGO | *1 ml PBS/FSGO | ||
*100 ul normal serum of same species as secondary antibody | |||
100 ul normal serum of same species as secondary antibody | |||
4 drops avidin blocking solution (Vector Labs) | 4 drops avidin blocking solution (Vector Labs) | ||
Invert tube to mix | *Invert tube to mix | ||
'''5.''' Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST | '''5.''' Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST | ||
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'''7.''' Aspirate off blocking solution | '''7.''' Aspirate off blocking solution | ||
'''8.''' Add primary antibody to vessels on slides. For 4 slides: antibody | '''8.''' Add primary antibody to vessels on slides. | ||
PBS/FSGO 1 ml | |||
normal serum 100 ul | For 4 slides: antibody | ||
biotin blocking solution (Vector Labs) 4 drops | *PBS/FSGO 1 ml | ||
*normal serum 100 ul | |||
*biotin blocking solution (Vector Labs) 4 drops | |||
'''9.''' Incubate 4 oC overnight in humidified chamber. | '''9.''' Incubate 4 oC overnight in humidified chamber. | ||
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'''1.''' Aspirate primary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate | '''1.''' Aspirate primary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate | ||
'''2.''' Make up secondary antibody: | '''2.''' Make up secondary antibody: | ||
per ml: | per ml: | ||
5 ul secondary antibody | *5 ul secondary antibody | ||
100 ul normal serum | *100 ul normal serum | ||
1 ml FSGO/PBS | *1 ml FSGO/PBS | ||
'''3.''' Blot slides with kimwipe and apply secondary antibody; incubate in humidified chamber 1 hour, room temperature | '''3.''' Blot slides with kimwipe and apply secondary antibody; incubate in humidified chamber 1 hour, room temperature | ||
| Line 83: | Line 86: | ||
'''4.''' During incubation, make up ABC solution from kit: | '''4.''' During incubation, make up ABC solution from kit: | ||
per ml: | per ml: | ||
1 ml plain PBS | *1 ml plain PBS | ||
20 ul A solution | *20 ul A solution | ||
20 ul B solution | *20 ul B solution | ||
Allow to rock at room temperature for at least 30 min in order to | Allow to rock at room temperature for at least 30 min in order to form complex | ||
form complex | |||
'''5.''' Aspirate secondary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate | '''5.''' Aspirate secondary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate | ||
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'''7.''' Make up DAB solution: | '''7.''' Make up DAB solution: | ||
Approximately 30 min before using; add H2 O2 just before use | Approximately 30 min before using; add H2 O2 just before use | ||
10 ml plain PBS | *10 ml plain PBS | ||
1 DAB tablet | *1 DAB tablet | ||
7.5 ul H2 O2 (30%) | *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. | Test DAB and ABC by adding small amount of DAB to a small amount of left over ABC, should turn dark brown immediately. | ||
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'''14.''' Dehydrate, 5 minutes each: | '''14.''' Dehydrate, 5 minutes each: | ||
3 x absolute ETOH | *3 x absolute ETOH | ||
2 x xylene | *2 x xylene | ||
'''15.''' Coverslip | '''15.''' Coverslip | ||
Revision as of 10:10, 16 September 2008
Immunohistochemistry Protocol
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. Destroy endogenous peroxidase activity
200 ml methanol + 3 ml (30% H2O2) in slide tub
- make just before use, Incubate 30 minutes, RT
2a. wash H2 O 5 min room temp.
1. Make up unmasking solution (Vector Labs; 4oC)
- 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
- 11
- 7
- 3
- 1
1. Cool slides in tub, covered, at room temp for 1 hour
2. During 1 hour, make up 0.5% FSGO 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.
Day 2:
1. Aspirate primary antibody solution and wash in FSGO/PBS in tub for 5 min at room temperature; agitate 2. Make up secondary antibody:
per ml:
- 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
15. Coverslip
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.
