Griffin:Immunohistochemistry Paraffin

• Korey Griffin Resource List

On The Napkin

• 1)Bake slides 1 hr, rehydrate xylene and EtOH series.
• 2)Antigen retrieve heat @96C (steam) 30-45 min. Wash TBST.
• 3)Sequenza or (semi automated) block sections 5% (donkey) serum.
• 4)Add primary (1:10-???) in 5% serum. Stain overnight @4C. Wash TBST. 3% H2O2 @ 15 min. Wash TBST.
• 5)Add secondary (1:100 HRP/Biotin) 1 hr. Wash. DAB detection

Tissue Structure(s)

• Bladder
• Intestine
• Stroma (cells performing function of the tissue or organ)
• Stroma
• Lumen/Lumenal
• Parenchyma
• Mesenchyme (Lamina Propria)

Histology Ig labeling / Signal Enhancement

• IHC Signal Substrate Strategies
• Methodology
• Polymer IHC
• Chromogenic Substrates

Tyramide Detection Amplification

Horseradish peroxidase (HRP) catalyzes covalent deposition and binding of fluorochrome linked tyramide substrates onto target protein or nucleic acid sequence in situ.

• Tyramide signal amplification (TSA)
• CST tutorial

Frozen Tissue

Fresh fixed (protein (antibody) labeling)

• Perfusion (blood removal)/ PFA /Sucrose/OCT/Cryosection (~7-20uM)
• 4% PFA /30% sucrose in 1xPBS (4C) until tissue sinks/ slice on cryostat / on-slide IHC / (optional) HIER 10mM citrate buffer, pH6.0.
• Post fixative = 30% sucrose/ w 70% fixative solution

Fresh non-fixed (RNA)

• No perfusion/fresh-frozen/Sucrose/OCT/Cryosection (~7-10uM)
• for protein labeling : HIER incompatible. Consider enzymatic and/or detergent treatment
• for protein labeling : 4% PFA (post-fixation)

Sucrose

• Cryopreservation (dehydration/4C) with sucrose (ie 30% sucrose/ w or w/out 70% fixative solution) is performed on fixed tissue prior to OCT embedding.
• Can be performed in a sucrose gradient of 15% sucrose until tissue sinks, then 25% sucrose until tissue sinks, then 30% sucrose until tissue sinks.
• Sucrose cryopreservative (antifreeze) protects tissue microstructure (ie protein) from damage/ prevents ice crystal formation in tissues when H2O freezes and expands, prevents freeze artifact or loss of tissue architecture.
• 30% sucrose tissue may take 3+ days to sink.
• perform in a 24-well plate.

OCT

• Optimal cutting temperature compound (OCT) cryo embedding matrix for cryostat sectioning of tissue specimens < -10 °C.
• Dry ice (pulverized) and store tissue at -80C until cutting.
• OCT medium is water soluble can remove w/ water or PBS (ie 1xPBS 20 minutes).

Immunohistochemistry Paraffin Section Protocol

Granular CD68 localization within macrophages of arterial athersclerotic plaque

Mature soluble MUC2 in human small intestine and colon in goblet cells

Mouse pancreas tissue; Red: Amylase in zymogengranules of acinar cells, and secreted amylase in ducts, Blue: DAPI (nuclear)

Immunohistochemistry determines protein expression profiles in tissues using chromagenic substrate (3,3′-Diaminobenzidine (DAB), benzene derivative). Primary antibody binds to the protein in tissue following with a conjugated peroxidase enzyme. Addition of hydrogen peroxide + DAB, catalyzes DAB to its oxidized, brown precipitate form.

The procedure below has been proven to be effective for labeling of PECAM-1, ICAM and VCAM using validated antibodies.

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

Quench endogenous peroxidase activity

200 ml methanol + 3 ml (30% H2O2) in slide tub

• make just before use, Incubate 30 minutes, RT

• wash H2O 5 min room temp.

Antigen Retrieval

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

Blocking

Detergents

• Triton-X and Tween-20 (<0.3% Triton X-100) are non-selective interact with protein and lipids creating pores and/or removing them from the membrane.

1. Cool slides in tub, covered, at room temp for 1 hour, then transfer to tub with plain room temperature PBS, 5 min

• 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)

2. 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

3. Circle vessels with ‘pap pen’ ALWAYS KEEP SLIDES MOIST

4. Add blocking solution within circles; 1 hour in humidified chamber at room temperature, then aspirate off blocking solution

Primary Antibody

5. Add primary antibody @ 1:50 to vessels on slides.

For 4 slides: antibody

• PBS/FSGO 1 ml
• normal serum 100 ul
• OPTIONAL : biotin blocking solution (Vector Labs) 4 drops

6. Incubate 4 oC overnight in humidified chamber.

Secondary Antibody

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 at 1:100-1:500:

(1:200) 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

Substrate

• Enzyme x chromogenic substrate combinations

3,3′-Diaminobenzidine (DAB)

• Primary antibody bound tissue protein + peroxidase (Horseradish peroxidase (HRP) 40kDa) conjugated secondary antibody (or biotin linked secondary antibody + avidin-HRP). In the presence of hydrogen peroxide (H2O2 (oxidizing agent)),HRP dependent DAB oxidation yields a brown precipitate. Therefore protein localization is measurable using DAB as a substrate.
• DAB is water soluble in an unoxidized form / forms water-insoluble brown precipitate under oxidation (H2O2 + HRP).
• HRP and H2O2 form a complex in the presence of DAB; HRP catalyzes breakdown of H2O2 (oxidizing agent) into water (H2O) and oxygen (O2). DAB is oxidized during this process while providing an electron source to drive the reaction.
• HRP forms a complex with hydrogen peroxide and yields water and oxygen. Free oxygen reacts with DAB to generate brown precipitate.

DAB reaction

H2O2→HRP→O2+H2O

O2+DAB→colored precipitate

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 H2O2 (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

Counterstain

In filtered (#1 Whatman filter paper) 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.

Blueing

If needed for 1 min; wash in tap water

• Acid alcohol: 1% HCl in 50% EtOH
• Bluing reagent: commercially available (Fisher, VWR, Sigma (S5134), etc)

Blueing Reagents

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).

0.1% Sodium Bicarbonate

• Sodium bicarbonate --------------------------- 1 g
• Distilled water --------------------------------- 1000 ml

Mix to dissolve. The pH will be around 8.0 Store this solution at room temperature.

Note: this solution worked better than ammonia water solution according to testing result. Bluing for 30 seconds to 1 minute after hematoxylin staining and clearing/differentiation.

0.2% Ammonia Water Solution (Bluing)

• Ammonium hydroxide (concentrated) ------ 2 ml
• Distilled water -------------------------------- 1000 ml

Mix well. The pH will be around 10.0 Store this solution at room temperature.

Note: this solution is not as good as sodium bicarbonate according to testing result. Bluing for 30 seconds to 1 minute after hematoxylin staining and clearing/differentiation.

Lithium Carbonate Solution (Saturated)

• Lithium carbonate ---------------------------- 1.54 g
• Distilled water -------------------------------- 100 ml

Mix to dissolve and store at room temperature. Bluing for 30 seconds to 1 minute after hematoxylin staining and clearing/differentiation.

Dehydrate

5 minutes each:

• 3 x absolute ETOH
• 2 x xylene

Coverslip

Mount

Abbreviated Immunohistochemistry Protocol

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

DAB enhancement

Nickel

• Addition of HRP+ 3,3′-diaminobenzidine (DAB)+H2O2 deposits dark brown precipitate on the tissue. Addition of heavy metals (Nickel) the reaction product can become darker and more easily detectable.

DAB (0.5 mg/ml) with 0.08% nickel sulfate and 3% H2O2

• Volume total: 1 ml
• 2% nickel sulfate 40 ul
• DAB Solution 1 ml
• 30% H2O2 10 ul

To make: 2% Nickel sulfate, NiSO4.6H2O: 0.9 g into 45 ml E-pure dd H2O = 2% solution. Keep in Fridge. (Dilute: 1/25 to give a 0.08% solution.)

NOTE: Time to stain will be more rapid. Nickel solution develops more precipitate. Blue-black ppt

Copper

• 0.4% cupric sulfate (CuSO4 5H2O)In 0.9% NaCl

After DAB reaction has been completed and rinsed, immerse slides 5-20 mins, and then rinse with PBS. Darkens regular DAB color.Room temp cupric sulfate in a glass bottle is stable for many years.

Trouble Shooting

Precipitate in DAB substrate preparation (or on tissue)

DAB substrate preparation

• 3,3'-Diaminobenzidine (DAB)
• dH20
• Substrate Buffer
• Peroxidase Substrate (H2O2)

The DAB substrate preparation is not stable in solution for prolonged periods of time, and will precipitate in aqueous solution exposed to oxygen. A quick spin down of the DAB substrate preparation (3 minutes @ 10,000 g) prior to dropping on the slides will ensure only the aqueous fraction is used; in case there is any precipitate in the DAB substrate preparation, it will be pelleted by centrifugation.

Desired staining can take 5-15 minutes. The DAB substrate produces a dark brown precipitate in the presence of horseradish peroxidase. Any suspended precipitate or sediments that form during this step may be removed after the desired color has been achieved by several washes with dH2O.

Peroxide-based chromogenic assays tend to have hydroperoxides and chromogenic electron donors to react before exposure to HRP catalytic enzyme. This oxidation "background" effect requires that the Peroxidase Substrate solution be used immediately (no later than few hours). One factor contributing to oxidation is contamination of chromogen solutions with trace amounts of oxidizing agents such as transition metals.

DAB has a molecular weight of 214.1 and yields a brown precipitate in the presence of HRP and peroxide. The brown, insoluble product can be readily chelated with osmium tetroxide. This property makes DAB ideal for electron microscopy. The color produced by DAB can be intensified with the addition of metals such as nickel, copper, silver and cobalt that form complexes. The color produced by the metal complexes is darker than the color produced by DAB alone, enhancing the sensitivity in staining applications. Peroxide must be added to a substrate for colorimetric detection with HRP.

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

Avidin-Biotin

Streptavidin-Biotin

Both Avidin and Streptavidin are suitable for binding to secondary biotin-conjugated antibodies. Streptavidin lacks carbohydrate modification and has a near-neutral pI, so it can produce lower nonspecific binding than avidin. However deglycosylated avidin (Neutravidin) is more comparable to the size, pI and specific binding of streptavidin.

• 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 protein isolated from cultures of Streptomyces avidinii with 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 may 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 (Kd~10e-15 M). The bond formation is rapid and is unaffected over a wide range of pH.

References

John M. Sanders B.S. Cardiovascular Research Center, University of Virginia Charlottesville, VA.

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 |