Categories
BLOG

dab intensifies

Griffin:Immunohistochemistry Paraffin

Contents

Immunohistochemistry Paraffin Section Protocol

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

2 x absolute ETOH

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

  • 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

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:

  • 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

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:

  • 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

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

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

  • 3 x absolute ETOH
  • 2 x xylene

Coverslip

Abbreviated Immunohistochemistry Protocol

1. Deparaffinize and rehydrate 5 min tray

2 x absolute ETOH

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 2×10 seconds, then 100% ethanol 2×10 seconds, then xylenes 3×10 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.

Griffin:Immunohistochemistry Paraffin Contents Immunohistochemistry Paraffin Section Protocol Immunohistochemistry determines protein expression profiles in tissues using chromagenic