Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3102: Immunohistochemical (IHC) detection of human or rat IGFBPs and IGF-I, and rat IGF-II in formalin fixed, paraffin embedded human and rat tissue sections by horseradish peroxidase staining

This document details a horseradish peroxidase staining protocol for the detection of IGFBPs, IGF-I in paraffin sections of human and rat tissues and IGF-II in rat tissue using Novozymes GroPep's Antibodies for Immunohistochemistry. In this protocol, the paraffin-embedded tissue section is first de-paraffinized, re-hydrated and then incubated with the appropriate Novozymes GroPep Antibodies for Immunohistochemistry primary antibody, followed by a biotinylated anti-rabbit immunoglobulin (secondary antibody). The specifically bound secondary antibody is then visualized by strepavidin-conjugated horseradish peroxidase.

Novozymes GroPep's Antibodies for Immunohistochemistry (IHC) have been tested in the appropriate human tissues and/or rat embryonic tissues. Pre-absorption of the antibody with 1-2 µg (100 µl of 10-20 µg/ml) of the corresponding Novozymes GroPep antigen (IGF-I, IGF-II or IGFBP) for 1 hour at room temperature abolishes the immunohistochemical staining.

Equipment and Reagents Required:

Equipment:

1. Solvent tanks
2. Slide racks to fit into the solvent tanks
3. Drying oven
4. Microwave oven
5. Humidified chamber or airtight plastic container
6. "Super Frost" microscope slides (Menzel-Glaser)
7. Coverslips
8. Pipettes and other general laboratory equipment
9. Light microscope

Novozymes GroPep's Antibodies for Immunohistochemistry and corresponding antigens:

A. Antibodies:

Anti-IGF-I:
IGF-I Antibody for Immunohistochemistry (Product Code: PABCa); 200 µg; Final dilution 1/50 to 1/200).

Anti-rat IGF-II:
IGF-II Antibody for Immunohistochemistry (Product Code: PAAL1); 50 µg; Final dilution 1/100).

Anti-IGFBPs:
IGFBP-1 Antibody for Immunohistochemistry (Product Code PAAH1; 20 µg; Final dilution 1/125 to 1/250).
IGFBP-2 Antibody for Immunohistochemistry (Product Code PAAI1; 20 µg; Final dilution 1/250 to 1/500).
IGFBP-3 Antibody for Immunohistochemistry (Product Code AAJ1; 20 µg; Final dilution 1/250 to 1/500).
IGFBP-4 Antibody for Immunohistochemistry (Product Code PAAG1; 20 µg; Final dilution 1/125 to 1/250).
IGFBP-5 Antibody for Immunohistochemistry (Product Code PAAF1; 20 µg; Final dilution 1/50 to 1/100).
IGFBP-6 Antibody for Immunohistochemistry (Product Code PAAE1; 20 µg; Final dilution 1/250 to 1/500).
IGFBP-rP1 Antibody for Immunohistochemistry (Product Code PAAD1; 20 µg; Final dilution 1/250 to 1/500).

Specificity of staining can be checked by blocking with 1- 2 µg of the corresponding antigen (100 µl of 10-20 µg/ml) for 1 hour at room temperature.

B. Antigens:

IGF-I:
For the IGF-I Antibody for Immunohistochemistry (Product Code: PABCa) we recommend using Novozymes GroPep LONG®R³IGF-I (Media Grade) (Product Code: AU200) as a competitive control.
LONG®R³IGF-I is an analog of human IGF-I that has a very low affinity for IGF Binding Proteins.

Rat IGF-II:
Rat IGF-II (Product Code: AAU020, 20 µg oR AAU100, 100 µg)

IGFBPs:
Human IGFBP-1 (Product Code BP1BU020, 20 µg)
Human IGFBP-2 (Product Code BP2BU020, 20 µg)
Human IGFBP-3 (Product Code BP3BU020, 20 µg)
Human IGFBP-4 (Product Code BP4BU020, 20 µg) or Rat IGFBP-4 (Product Code BP4CU015, 15 µg)
Human IGFBP-5 (Product Code BP5BU020, 20 µg) or Mouse IGFBP-5 (Product Code: BP5DU020, 20 µg) or Rat IGFBP-5 (Product Code: BP5CU020, 20 µg)
Human IGFBP-6 (Product Code BP6BU020, 20 µg)


Other Reagents:

1. Xylene
2. Absolute ethanol
3. Absolute methanol
4. Distilled or Milli-Q water
5. EDTA Solution, pH 8.0 (Zymed Cat. No. 00-5500 (Concentrate) or Cat. No. 00-5501 (Ready-to-Use))
6. 30 % (v/v) Hydrogen peroxide (H₂O₂).
7. 50 mM Tris-buffered saline (TBS), pH 7.8 at 22°C (Optional: 0.05 % (v/v) Tween 20), 150 mM NaCl OR OPTIMAX Wash Buffer (Biogenics Cat. No. HK 583-5K)
8. HISTOSTAIN®-PLUS Bulk Kit (Broad Spectrum, HRP) (Zymed Cat. No. 85-8943)
9. Horseradish peroxidase substrate / chromagen reagent: Aminoethyl Carbazole (AEC) (red) Substrate Kit (Zymed Cat. No. 00-2007)
10. Hydromount® mounting media (AGTC Bioproducts Ltd. Hydromount Cat. No. HS-106)


Protocol:

All procedures are carried out at room temperature unless otherwise stated.

A. Tissue section preparation:

1. Select the tissue sections desired for immunohistochemistry.

2. Prepare 5 µm sections of the paraffin-embedded tissues and mount on "Super Frost" slides.

3. Incubate the slides at 60°C for 60 minutes in an oven.

4. De-paraffinize the sections by placing the slides in a slide rack and immersing in xylene for 10 minutes.

5. Re-hydrate the sections by sequential washing in 100 % ethanol (twice), 95 % ethanol (twice) and distilled or Milli-Q water (three times) for 2 minutes each wash.

6. Sections are immersed in EDTA solution, pH 8.0 and heated in a microwave oven for 10 minutes at 736 Watts (92 % of 800 Watts). (Heat-induced epitope retrieval step).

7. Allow the sections to cool for 10 minutes.

8. Wash the sections three times in distilled or Milli-Q water for 2 minutes each wash.

9. Quench endogenous peroxidases by immersing the sections in 3 % (v/v) hydrogen peroxide (H₂O₂) in methanol for 5 minutes. This step is to reduce background and non-specific staining.

10. Wash the sections three times in distilled or Milli-Q water for 2 minutes each wash.


General Notes:

All procedures are carried out at room temperature unless otherwise stated.

A humidified chamber is required for some of the incubation steps.

Wipe slides around sections to remove most of the liquid from tissues before each incubation step.

Avoid drying of sections between steps.

Use sufficient reagent (~50 - 70 µl) to cover the sections for blocking or antibody incubations. Use 3-5 mls for each washing step.


B. Treatment with Primary Antibody:

1. Dissolve the appropriate Novozymes GroPep Antibody for Immunohistochemistry antibody in sterile distilled or Milli-Q water in accordance with the directions on the Certificate of Analysis supplied with the product (Stock Solution).

Dispense into convenient aliquots and store at - 20°C.

Avoid repeated freeze-thawing of the diluted antibody.

2. Prepare a Working Solution by diluting the Stock Solution in Antibody Dilution Reagent (Zymed Cat. No. 00-3118) and mix thoroughly. For use we recommend diluting the Stock Solution a further:

IGF-I Antibody for Immunohistochemistry (Product Code: PABCa): 1: 50
IGF-II Antibody for Immunohistochemistry (Product Code: PAAL1): 1:100
IGFBP Antibodies for Immunohistochemistry (Product Codes: PAAD1, PAAE1, PAAF1, PAAG1, PAAH1, PAAI1 or PAAJ1): 1:12.5 to 1:25

(See list of recommended final dilutions above). Prepare the Working Solution fresh each time.

3. Perform a Serum Blocking step for 20 minutes using 100 µl for each section (or sufficient reagent to cover the sections) of ready-to-use Reagent A in the HISTOSTAIN®-PLUS Bulk Kit (Broad Spectrum, HRP) (Zymed Cat. No. 85-8943).

4. Agitate sections, pour off excess liquid and blot around edges. DO NOT RINSE.

5. Incubate each section with 50 - 70 µl (or sufficient reagent to cover the sections) of the Primary Antibody Working Solution for 60 minutes in a humidified chamber.

6. Sections washed three times with TBS or OPTIMAX Wash Buffer for 5 minutes each wash.


C. Treatment with Secondary Antibody:

1. Incubate each section with the biotinylated secondary antibody using 100 µl (or sufficient reagent to cover the sections) of ready-to-use Reagent B in the HISTOSTAIN®-PLUS Bulk Kit (Broad Spectrum, HRP) (Zymed Cat. No. 85-8943) for 30 minutes in a humidified chamber.

2. Sections washed three times with TBS or OPTIMAX Wash Buffer for 5 minutes each wash.


D. Detection:

1. Incubate each section with the enzyme conjugated horseradish peroxidase - strepavidin using 100 µl (or sufficient reagent to cover the sections) of ready-to-use Reagent C in the HISTOSTAIN®-PLUS Bulk Kit (Broad Spectrum, HRP) (Zymed Cat. No. 85-8943) for 30 minutes in a humidified chamber.

2. Sections washed three times with TBS or OPTIMAX Wash Buffer for 5 minutes each wash.

3. Incubate the sections with horseradish peroxidase substrate / chromagen reagent: Aminoethyl Carbazole (AEC) (red) Substrate Kit (Zymed Cat. No. 00-2007) in the dark for 10 minutes in a humidified chamber, in accordance with the manufacturers recommendations.

4. Sections washed three times with distilled or Milli-Q water for 5 minutes each wash.

5. Sections stained with hematoxylin for 1 - 2 minutes.

6. Sections washed three times with distilled or Milli-Q water for 5 minutes each wash.

7. Blot around edges of each section and mount using Hydromount® mounting media (AGTC Bioproducts Ltd. Hydromount Order No. HS-106).

8. Examine using a light microscope. Immunohistochemically labelled areas will exhibit a red-brown staining.

This method, together with the immunohistochemistry pictures of human and rat tissues, were kindly supplied by Prof. Zerem Freier, Shaare Zedek Medical Center, Jerusalem, Israel.


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