Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3051: Immunohistochemical (IHC) Detection of IGF-I in paraffin embedded sections of rat tissues by Immunoperoxidase staining

Introduction

Insulin-like Growth Factor (IGF-I) is produced by most tissues in the body. Immunohistochemical detection of IGF-I is one of the important tools for studying the local expression of IGF-I peptide in tissues. This document details the immunoperoxidase staining protocol for IGF-I detection in paraffin sections using Novozymes GroPep's IGF-I Antiserum for Immunohistochemistry (Product Code PABCa). In this protocol, the paraffin-embedded tissue section is first incubated with Novozymes GroPep's primary antibody, then with a biotinylated anti-rabbit IgG antibody (secondary antibody). The specifically bound secondary antibody is then visualised by a pre-formed avidin-biotinylated horseradish peroxidase complex and substrate. This method has been called the ABC technique, and is currently the most sensitive method for immunoperoxidase staining.

Novozymes GroPep's IGF-I Antiserum for immunohistochemistry (Product Code PABCa) has been used for human, rat and fish tissues, and as bovine and porcine IGF-I have the same amino acid sequence as human IGF-I, we anticipate this antiserum would be suitable for immunohistochemistry studies in these tissues.

Importantly, as human IGF-II has less than 0.1% cross-reactivity with Novozymes GroPep's IGF-I Antiserum, only IGF-I will be detected. Pre-absorption of the antiserum (diluted 1/50) with 10 µg (100 µl of 100 µg/ml) human LONG®R³IGF-I at room temperature for 2 hours abolishes the immunohistochemical staining in human skin and rat 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. Pipettes and other general laboratory equipment

6. Horizontal shaker

7. Light microscope


Reagents

1. Tris-buffered saline (TBS): 10 mM Tris, pH 7.4, 150 mM NaCl.
To make 4 litres, add ~1.5 litres 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 7.4 using concentrated HCl. Make up to a final volume of 4 litre with distilled or Milli-Q water in a 4 litre volumetric flask.

2. Endogenous quenching solution : Add 2 ml 30 % (v/v) hydrogen peroxide solution and 0.1 g sodium azide to 100 ml of TBS.

3. Blocking Solution (4 %) : Dissolve 4 g skim milk powder in 100 ml TBS with gentle stirring. Make up fresh each day.

4. 1 % (w/v) BSA/TBS: Dissolve 1 g bovine serum albumin (BSA) (Sigma Catalog #A7888) in 100 ml TBS with gentle stirring. Use this solution to dilute antibodies and horse serum.

5. Dissolve Novozymes GroPep’s IGF-I Antiserum, affinity purified for Immunohistochemistry (Product Code PABCa) in 200 µl 1 % BSA/TBS. Make up dilutions of the antisera in 1 % BSA/TBS as required, and mix thoroughly. Dispense the remaining antisera into convenient aliquots and store at - 20°C. Avoid repeated freeze-thawing of the diluted antiserum.

6. ABC kit: Vectastain Universal Elite Kit (Vecta Catalog #PK6200 (Immunodiagnostics P/L Aust))

7. DAB solution: Zymed’s DAB (3,3'-diaminobenzidine tetrahydrochloride) solution (Liquid DAB Plus, Catalog No #00-2020, (Bioscientific, Aust)).

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 liver can be used as positive controls. We routinely use 3 - 6 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 % ethanol for 5 minutes, then with 80 %, 50 %, 30 % (all v/v) ethanol for 5 minutes each.

4. Soak sections in distilled water for 5 minutes.

5. Incubate for 10 minutes in hydrogen peroxide - azide solution to quench any endogenous peroxidase activity.

6. Wash sections with TBS with gentle shaking, three times for 5 minutes.

7. Carefully wipe around the sections to absorb excess moisture.

8. Incubate sections with blocking solution (4 % in TBS) for 20 minutes.

9. Wash sections twice with TBS with gentle shaking for 5 minutes.

10. Incubate sections with horse serum (1/100 dilution in 1 % BSA / TBS) for 20 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. A normal horse serum is specified here since a horse anti-rabbit secondary antibody was used.

11. Tap off blocking serum. Do not wash sections.

12. Place sufficient of the Novozymes GroPep diluted primary antibody solution or negative control to completely cover the sections, place in a humidified container and incubate at room temperature for 1 hour or at 4°C overnight. To establish IGF-I staining intensity the first time, we suggest trying a 1/50 to 1/100 dilution in 1 % BSA/TBS of Novozymes GroPep's IGF-I antiserum for Immunohistochemistry (Product Code PABCa).

13. Wash sections with TBS with gentle shaking, three times for 5 minutes.

14. Incubate with biotinylated universal secondary antibody from the Vectastain Universal Elite Kit (#PK6200) for 30 min at room temperature in a humidified container. (Dilute 20 µl secondary antibody plus 20 µl horse serum 1/100 in 2 ml 1 % BSA/TBS to use for incubation).

15. Wash sections with TBS with gentle shaking, three times for 5 minutes.

16. Incubate sections with ABC complex for 30 minutes. Make up this ABC complex at least 30 minutes prior to use.

17. Wash sections with TBS with gentle shaking, three times for 5 minutes.

18. 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 rat 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.

19. Rinse slides gently with tap water to wash off DAB solution, then rinse with TBS.

20. Lightly counterstain with Mayer's haematoxylin (Sigma, Catalog #MHS-1) for 1-5 minutes. The optimal concentration and staining time should be determined for different tissues and whether using fresh or old solution.

21. Wash sections briefly with 2 changes of tap water.

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

23. Mount coverslip using Depex mounting medium.

24. Examine by light microscopy. This protocol should generate brown IGF-I staining with a pale blue counterstained background.


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