Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3050: Immunohistochemical (IHC) Detection of human or rat IGF-I in OCT sections by Extravidin - Cy3 staining

Introduction

Insulin-like Growth Factor-I (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 a Streptavidin-Cy3 protocol for IGF-I detection in TissueTek O.C.T. frozen tissue sections using Novozymes GroPep's IGF-I Affinity Purified Antibody for Immunohistochemistry, (Product Code: PABCa). In this protocol, the frozen sections are incubated first with this antibody (primary antibody), then with a biotinylated anti-rabbit IgG antibody (secondary antibody). The specifically bound secondary antibody is then visualised with the fluorescent streptavidin-Cy3.

Novozymes GroPep's IGF-I Antbody 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 would anticipate this antiserum to be suitable for immunohistochemistry studies in these tissues.

Importantly, as Novozymes GroPep's IGF-I Antibody for Immunohistochemistry has less than 0.1% cross-reactivity with human IGF-II, only IGF-I will be detected. Pre-absorption of the antiserum (diluted 1/500) with 50 µg/ml human LONG®R³IGF-I at room temperature for 30 minutes abolishes the immunohistochemical staining in human and rat skin sections.


Novozymes GroPep Reagents

Store lyophilized reagents at 2-8°C.

Primary Antibody (Novozymes GroPep IGF-I Antibody for Immunohistochemistry, Affinity Purified, Product Code: PABCa) Dissolve the contents of the vial in 200 µl of sterile distilled or Milli-Q water. Aliquot and freeze at -20°C. Dilute in PBS (see Wash Buffer below).

Novozymes GroPep's LONG®R³IGF-I (Media Grade) (Catalog Code: AU100; 100 µg)

Reagents and Equipment Required

Suppliers indicated are given as examples. Each laboratory should obtain their own reagents and validate the assay

1. TissueTek O.C.T. (Bayer Corporation, Pittsburgh, PA, USA; Catalog Code: 4583)

2. Cy3 Streptavidin (Sigma Chemical Co., St Louis, MO, USA; Catalog Code: S6042)

3. Secondary Antibody - Biotinylated sheep anti-rabbit IgG (Sigma Chemical Co., St Louis, MO, USA; Catalog Code: B9140)

4. Shandon Immuno-Mount (Life Sciences Intl, Cheshire, UK; Available through Jomar Diagnostics, Adelaide, Australia; Catalog Code: 9990402)

5. Wash Buffer - phosphate buffered saline (PBS) : 10 mM Phosphate buffer, pH 7.4, 150 mM NaCl


Comments

1. TissueTek O.C.T frozen sections are preferred.
Paraformaldehyde fixed and paraffin embedded sections are found to have a high background
fluorescence. See protocols suitable for paraffin embedded sections using the sensitive immunoperoxidase (ABC) detection system.

2. It is not necessary to block with either 10 % BSA or milk powder when using Novozymes GroPep’s IGF-I Antbody
for Immunohistochemistry.

3. LONG®R³IGF-I competition for antibody binding can be used to show specificity of staining for IGF-I.


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 the slide before the next incubation. Avoid drying of specimens between steps. For all incubations use sufficient reagent to cover the sections (50 - 100 µl).

1. Place the tissue in TissueTek O.C.T. and then freeze in liquid nitrogen. Store tissue at –80°C. Cut 7 mm sections of the frozen O.C.T. tissue and fix the section in acetone for 20 min at room temperature.

2. Rinse sections 3-4 times in PBS.

3. Add 100 µl per section of the primary antibody, diluted (1/100 for human tissues; 1/200 for rat tissues) in PBS. Incubate in a humidified chamber at 4°C overnight.

4. Rinse sections 3-4 times in PBS.

5. Add 100 µl per section of the secondary antibody, diluted 1/1000 in PBS. Incubate at room temperature for 1 hour.

6. Rinse sections 3-4 times in PBS.

7. Add 100 µl per section of Cy3 streptavidin, diluted 1/200 in PBS. Incubate at room temperature for 45 min.

8. Rinse sections 3-4 times in PBS.

9. Mount slides with Shandon Immuno-mount and coverslip.

10. View with a fluorescent microscope using a green filter.


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