Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #4110: Immunohistochemical (IHC) Detection of IGF-I in paraffin sections of Barramundi (fish) kidney 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 (Catalog 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 (Catalog 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 100 µg/ml human LONG®R³IGF-I (Media Grade) 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. Phosphate-buffered saline (PBS): 10 mM Phosphate buffer, pH 7.4, 150 mM NaCl.

2. Blocking Solution: Dissolve 1 g BSA in 80 ml PBS with gentle stirring. Mix in 20 ml normal swine serum. Make up fresh each day.

3. 1% (w/v) BSA/PBS: Dissolve 1 g bovine serum albumin (BSA) (Sigma Catalog #A7888) in 100 ml PBS with gentle stirring. Use this solution to dilute antibodies and control swine serum.

4. Dissolve Novozymes GroPep’s IGF-I Antiserum, affinity purified for Immunohistochemistry (Catalog Code PABCa) in 200 µl 1% BSA/PBS. Make up dilutions of the antisera in 1% BSA/PBS 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.

5. DAKO (Dako Co, Carpinteria, CA 93013, USA) swine anti-rabbit IgG horseradish peroxidase conjugate.

6. DAB solution: Sigma’s 3,3'-diaminobenzidine tetrahydrochloride (DAB) tablets (Catalog #D-5905). Dissolve a 10 mg DAB tablet in 15 ml of PBS and filter through a 0.22 µm filter. Add 12 µl of 30 % 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 liver can be used as positive controls. We routinely use 3 - 6 µm paraffin sections of Bouins or formalin (4 %) fixed tissue mounted on gelatin-coated glass slides.

2. Deparaffinise sections twice, 5 minutes each in 100 % xylene (BDH, Catalog #30575, Merck Pty Ltd, Kilsyth, Vic 3137, Australia).

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

4. Soak sections in distilled or Milli-Q water for 3 minutes.

5. Cover sections with fresh blocking solution. Leave at room temperature for 5 min then remove blocking solution.

6. Place sufficient of the diluted primary antibody solution or negative control to completely cover the sections, place in a humidified container and incubate at room temperature for 45 min. To establish IGF-I staining intensity the first time, we suggest trying a 1/200 to 1/400 dilution in 1% BSA/TBS of Novozymes GroPep’s IGF-I antiserum for Immunohistochemistry (Catalog Code PABCa).

7. Wash sections with PBS with gentle shaking, three times for 5 minutes.

8. Incubate slides in methanol containing 0.3 % hydrogen peroxide for 30 min room temperature.

9. Wash sections with PBS with gentle shaking, three times for 5 minutes.

10. Cover slides with DAKO anti-rabbit IgG horseradish peroxidase conjugate, diluted 1/300 to 1/600 with PBS. Incubate in humidified chamber at room temperature for 45 min.

11. Wash sections with PBS with gentle shaking, three times for 5 minutes.

12. Incubate in DAB solution for 5 min room temperature. The optimal DAB incubation time should be established for each kind of specimen. Check under the microscope for positive brown colour staining. Avoid long DAB incubation time to minimise the background staining.

13. Wash sections in 2 changes of tap water.

14. Counterstain as required. Wash sections in 2 changes of tap water.

15. 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.

16. Mount coverslip and examine by light microscopy. This protocol should generate brown IGF-I staining.


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