Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3510: Immunohistochemical (IHC) Detection of human EGF in Paraffin Sections by Immunoperoxidase Staining

Introduction

Epidermal Growth Factor (EGF) is produced mainly in the kidney and salivary gland, but is also found in duodenum, pancreas, thyroid gland and some carcinomas such as gastrointestinal carcinomas (J. Cancer Res. Clin. Oncol 116, 121-131). Immunohistochemical detection of EGF is one of the important tools for studying the local expression of EGF peptide in tissues. This method details the immunoperoxidase staining protocol for EGF detection in paraffin sections using Novozymes GroPep’s anti-human EGF polyclonal antiserum (Catalog Code PAD1). In this protocol, the paraffin-embedded tissue section is incubated first with this antiserum (primary antibody), then with a biotinylated anti-rabbit IgG antibody (secondary antibody). The specifically bound secondary antibody is then visualised by a preformed avidin-biotinylated horseradish peroxidase complex and 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate. This method has been called the ABC technique, and is currently the most sensitive method for immunoperoxidase staining.

Novozymes GroPep’s anti-human EGF polyclonal antiserum does not cross-react with human Transforming Growth Factor-alpha (TGF-α) or mouse EGF. Preabsorption of the antiserum (1/200) with 45 µg/ml human EGF at room temperature for 2 hours abolishes the immunohistochemical staining of human kidney sections.

Equipment and Reagents Required

Equipment

1. Solvent tanks

2. Slide racks to fit into the solvent tanks

3. Humidifying chamber or airtight plastic container

4. Coverslips

5. “DAKO” pen (Dako Co, Carpinteria, CA 93013, USA) (or equivalent)

6. Pipettes and other general laboratory equipment

7. Horizontal shaker

8. Light microscope


Reagents

1. Endogenous quenching solution : To prepare 300 ml of 1% (v/v) hydrogen peroxide, add 10ml of 30% (v/v) solution to 290 ml of distilled or Milli-Q water.

2. Tris-buffered saline (TBS): 150 mM NaCl, 10 mM Tris base, pH 7.5.
To make 4 litres, add ~1.5 litres of distilled or Milli-Q water to a 2 litre beaker. Add 4.84 g Tris base (Sigma, St Louis, MO, USA, catalog #T1503) and 35 g NaCl. Stir to dissolve. Adjust pH to 7.5 using concentrated HCl. Make up to a final volume of 4 litre with distilled or Milli-Q water in a 4 litre volumetric flask.

3. 2% (w/v) BSA/TBS: Dissolve 2 g bovine serum albumin (BSA) (Sigma catalog #A7888) in 100ml TBS with gentle stirring. Use this solution to dilute antibodies.

4. Novozymes GroPep’s anti-human EGF polyclonal antiserum (Catalog Code PAD1) diluted within the range of 1/100 to 1/600 with 2 % BSA/TBS. To make up 1/100 dilution from one vial lyophilized antiserum (equivalent to the original 5 µl serum), add 500 µl 2 % BSA/TBS and mix thoroughly. Dispense the surplus solution into convenient aliquots and store at - 20°C. Avoid repeated freeze-thawing of the diluted antiserum.

5. ABC complex: Dako’s ABC kit (Catalog #K0355). To make up: add 10 µl Solution A to 1 ml of TBS. Mix well, then add 10 µl of Solution B, mix and incubate for at least 30 minutes prior to use.

6. DAB solution: Sigma’s 3,3'-diaminobenzidine tetrahydrochloride (DAB) tablets (Catalog #D-5905). Dissolve a 10 mg DAB tablet in 16 ml of TBS and filter through a 0.22 µm filter.
Add 16 µl of 30 % (v/v) hydrogen peroxide just prior to use.

Protocol

Carry out all procedures at room temperature (unless otherwise specified). A humidified chamber is required for some of the incubation steps. Wipe slides around sections and remove most of the liquid from tissues before next incubation. Avoid drying of specimens between steps. Use 50 - 100 µl or sufficient reagent to cover the sections for blocking or antibody incubations.

1. Select the tissue sections desired for immunohistochemistry. Human kidney or salivary gland can be used as positive controls. We routinely use 3 - 5 µm paraffin sections mounted on gelatin-coated glass slides.

2. Deparaffinise sections twice, 10 minutes each in 100 % xylene (BDH, Catalog #30575, Merck Pty Ltd, Kilsyth, Vic 3137, Australia).

3. Hydrate sections with 100 %, 80 %, 50 %, 30 % (all v/v) ethanol for 5 minutes each.

4. Soak sections in distilled or Milli-Q water for 5 minutes.

5. Incubate for 30 minutes in 1 % (v/v) hydrogen peroxide to quench any endogenous peroxidase activity.

6. Rinse sections with gentle shaking for 5 minutes in distilled or Milli-Q water in a solvent tank, then wash in TBS with gentle shaking for 5 minutes.

7. Carefully wipe around the sections to absorb excess moisture. Using a “DAKO” or “PAP” pen draw on the glass slide making a complete circle around each section. This hydrophobic barrier will confine reagents onto the section thus preventing their spread.

8. Incubate sections with 5 % (v/v) blocking serum for 90 minutes to suppress non-specific binding of immunoglobulins. The host species of this normal serum should be the same as that for the secondary antibody. We routinely use normal pig serum since we use a pig anti-rabbit secondary antibody.

9. Tap off blocking serum, briefly rinse sections with TBS in a solvent tank.

10. Place sufficient primary antibody solution or negative control to completely cover the sections, place in a humidified container and incubate at 4°C overnight (around 16 hours) or at room temperature for 2 hours. Novozymes GroPep’s anti-human EGF polyclonal antiserum (Catalog Code PAD1) is recommended to be diluted at 1/100 to 1/600 in 2 % BSA/TBS. To establish EGF staining intensity the first time, we suggest trying a
1/100 dilution.

11. Wash sections with gentle shaking with: (1) 0.01 % (v/v) Tween-20 in TBS for 10 minutes; (2) Repeat (1);
(3) TBS for 10 minutes.

12. Incubate with biotinylated secondary antibody for 1 hour in a humidified container. We routinely use Dako’s biotinylated swine anti-rabbit IgG (Catalog #E0353) at 1/400 diluted in 2 % (w/v) BSA/TBS.

13. Wash with gentle shaking with: (1) 0.01 % (v/v) Tween-20 in TBS for 15 minutes; (2) TBS for 10 minutes.

14. Incubate sections for 30 minutes with ABC complex (made up at least 30 minutes prior to use).

15. Rinse sections briefly in TBS in a solvent tank then wash in TBS with gentle shaking for 5 minutes.

16. Incubate in DAB solution for 1 to 10 minutes. The optimal DAB incubation time should be established for each kind of specimen. We routinely use 2 minutes DAB reaction with human formalin-fixed and paraffin-embedded kidney sections. Check under microscope for positive brown colour staining. Avoid long DAB incubation time to minimise the background staining.

17. Rinse slides gently with tap water to wash off DAB solution.

18. Counterstain with 0.5 % (w/v) methyl green (Sigma, Catalog #M-8884) for 1 to 10 minutes. Determine the optimal concentration and staining time for different tissues and fresh or old methyl green solution.

19. Rinse with tap water briefly.

20. Dehydrate sections for 2 minutes each with a graded series of ethanol and then transfer to xylene :
30 % (v/v), 70 % (v/v), 100 % ethanol and 100 % xylene.

21. Mount coverslip using Histolabs’s colour fast Ultramount mounting medium (Histolabs, PO Box 127, Riverstone, NSW 2765, Australia).

22. Examine by light microscopy. This protocol should generate brown EGF staining with a green counterstained background.


© Novozymes GroPep Limited
28 Dalgleish Street
Thebarton SA 5031
PO Box 10065 BC
Adelaide SA AUSTRALIA
Telephone +61 (0)8 8354 7700
Facsimile +61 (0)8 8354 7788
Email: AUAD-bioreagents@novozymes.com
Website: www.gropep.com.au