Griffin:Immunohistochemistry Paraffin

Immunohistochemistry Protocol 1

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
• 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 (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.

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

• Acid alcohol: 1% HCl in 50% EtOH
• Bluing reagent: commercially available (Fisher, VWR, Sigma, etc)

Procedure: Dip slides quickly in acid alcohol and immediately wash with tap water (or squirt water on the slides) Dip slides briefly in Bluing Reagent. Wash slides with tap water and continue with the IHC staining procedure(i.e., dehydrate stained tissues and mount the slides).

14. Dehydrate, 5 minutes each:

• 3 x absolute ETOH
• 2 x xylene

15. Coverslip

1. Barringhaus KG, Phillips JW, Thatte JS, Sanders JM, Czarnik AC, Bennett DK, Ley KF, and Sarembock IJ. Alpha4beta1 integrin (VLA-4) blockade attenuates both early and late leukocyte recruitment and neointimal growth following carotid injury in apolipoprotein E (-/-) mice. J Vasc Res. 2004 May-Jun;41(3):252-60. DOI:10.1159/000078646 |
2. Manka D, Forlow SB, Sanders JM, Hurwitz D, Bennett DK, Green SA, Ley K, and Sarembock IJ. Critical role of platelet P-selectin in the response to arterial injury in apolipoprotein-E-deficient mice. Arterioscler Thromb Vasc Biol. 2004 Jun;24(6):1124-9. DOI:10.1161/01.ATV.0000127619.04687.f4 |

Immunohistochemistry Protocol 2

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

2. Antigen Retrieval

3. Rinse sections in 2 changes of PBS washing buffer in separate beakers, 2 minutes each.

4. Serum Blocking: incubate sections with 10% normal serum blocking solution for 30 minutes to block non-specific binding of immunoglobulin.

5. Primary Antibody: incubate sections with Primary antisera diluted 1:50 in 3% serum PBS, for 1 hour at room temperature to overnight 4C.

6. Rinse in PBS washing buffer for 2 x 2 min.

7. Secondary Antibody: incubate sections with conjugated secondary antibody 1:100-500 in 3% serum PBS, for 1 hour at room temperature.

8. Rinse in PBS washing buffer for 3 x 2 min.

9. Counter-stain in Gill´s formulation #2 hematoxylin for 5–10 seconds. Immediately wash with several changes of deionized H2O.

10. Dehydrate: Soak in 95% ethanol 2x10 seconds, then 100% ethanol 2x10 seconds, then xylenes 3x10 seconds. Pipet 1–2 (5ul) drops of permanent mounting medium, cover with a glass coverslip and observe by light microscopy.

Controls

• Chromogen only; no primary or secondary antibody
• Conjugate and chromogen control; no primary
• Biotinylated antibody, conjugate, chromogen control
• Isotype control ( for monoclonal antibodies only)
• Species control (ie. incubation with normal goat IgG)
• Absorption control

Preventing tissues from peeling off the slide

• Insufficient fixation or unfixed tissues tend to come off slides more easily. Fix tissues longer to get completely fixation of tissues.
• Formaldehyde fixed frozen sections are more prone to falling off slides. Try to dry slides for much longer time or use alternative fixation such as acetone or alcohol.
• Tissue sections tend to come off slides more often on regular slides (uncharged or uncoated). Always use positively charged or coated slides for immunostaining.
• You may just have had a bad batch of slides. Replace with a new batch of slides.
• The disposable blades have oil on them. Clean disposable blades with xylene.
• Wrinkles presented in the sections during the initial mounting. Try to spread the sections out and mount the sections on slides with wrinkle free.
• Paraffin sections may not be dried completely before placing in the oven. Allow paraffin sections to air dry at least 30 minutes before placing in the oven at 56 C overnight.
• Frozen sections may not be dried completely before fixation and immunostaining procedure. Allow frozen sections to air dry for at least 30 minutes before fixation and then air dry for another 30 minutes before immunostaining.
• Antigen retrieval procedure can cause sections to come off slides, especially when EDTA (pH8.0) or Tris-EDTA (pH9.0) such high pH antigen retrieval solution is used. Use low pH solution such as citrate buffer (pH6.0) antigen retrieval Solution to replace EDTA (pH8.0) or Tris-EDTA (pH9.0) retrieval solution if it is possible.
• Distilled water alone may make sections come off slides easy. Always use buffer solution to wash or rinse slides.
• Bone (especially the cartilage) tends to fall off slides after heat treatment. Try other alternative retrieval methods such as enzyme digestion.
• Antigen retrieval devices may be trouble. Try to use waterbath or steamer in stead of microwave or pressure cooker.
• Plain water in waterbath for mounting paraffin sections. Add some gelatin in waterbath for mounting paraffin sections.
• If you have tried everything above and the problem persists. Try gelatin coated slides and it has been working fairly well.

Avidin versus Streptavidin

Both Avidin and Streptavidin are suitable for binding to Biotin conjugated secondary biotin-conjugated antibodies.

• Avidin is a tetrameric protein originally isolated from chicken egg white with a MW of about 67K and an isoelectric point of about 10.
• Streptavidin is a biotin binding isolated from cultures of Streptomyces avidinii. It has Avidin-like binding characteristics; however, it has a MW of about 16K and an isoelectric point of 5-6.

One mole Avidin will bind four mole biotin. The high pI of Avidin can cause binding to acidic structures such as DNA. Biotin is a naturally occurring vitamin with a MW of 244.31 and an isoelectric point of 3.5. The Avidin-Biotin interaction is the strongest known non covalent, biological interaction (Ka=1015 M-1). The bond formation is rapid and is unaffected over wide range of pH.