Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3101: Immunohistochemical (IHC) Detection Of IGFBP-3 In Human Liver (OCT) Sections Using Extravidin - Cy3 Staining

Human Insulin-Like Growth Factor Binding Protein-3 (hIGFBP-3) is a 264 amino acid protein that is one of the six classical IGF binding proteins that play an important role in stabilizing and regulating the activity of IGFs in vivo. Human IGFBP-3 is a 40-44 kDa glycoprotein that is the most abundant IGFBP in serum and milk. It is produced by non-parenchymal hepatic cells and circulates in serum, binding IGF-I or IGF-II in conjunction with an acid labile subunit (ALS) to form a 150 kDa circulating complex at a serum concentration of about 100 nM. Free IGF-I has a half-life of ~10 min in serum. This can be increased to ~25 min if bound to IGFBP-3 and to ~15 h if complexed with IGFBP-3 and ALS (Rechler, MM & Clemmons, DR (1998) TEM 9, 176-183).

This document details the ExtrAvidin-Cy3 staining protocol for IGFBP-3 detection in human liver (OCT) sections using Novozymes GroPep's IGFBP-3 Antibody for Immunohistochemistry (Catalog Code PAAJ1). In this protocol, the tissue sections are incubated with Novozymes GroPep's primary antibody, followed by a biotinylated anti-rabbit immunoglobulin (secondary antibody). The specifically bound secondary antibody is then visualized by ExtrAvidin conjugated Cy3.

Novozymes GroPep's IGFBP-3 antibody for immunohistochemistry (Catalog Code PAAJ1) has been tested in human liver and paraffin sections of rat embryonic tissues. Pre-absorption of the antibody (diluted 1/500 to 1/1,000) with 2 µg (100 µl of 20 µg/ml) of human IGFBP-3 (Novozymes GroPep Catalog Code BP3BU020) at room temperature for 1 hour abolishes the immunohistochemical staining.


Equipment and Reagents Required

Equipment

1. Solvent tanks
2. Slide racks to fit into the solvent tanks
3. Humidified chamber or airtight plastic container
4. Gelatin-coated glass slides.
5. Coverslips
6. Pipettes and other general laboratory equipment
7. Horizontal shaker
8. UV microscope

Reagents

1. Dissolve Novozymes GroPep's IGFBP-3 Antibody for Immunohistochemistry (Code PAAJ1; 20 µg) in 400 µl sterile distilled or Milli-Q water (a 1/20 dilution). Make up dilutions of the antibody (final antibody dilution 1/500 to 1/1,000) in 1% BSA/PBS as required, and mix thoroughly. Dispense the remaining antibody (1/20 dilution) into convenient aliquots and store at - 20°C.

Avoid repeated freeze-thawing of the diluted antibody.

2. TissueTek O.C.T. (Bayer Corporation #4583)

3. Biotinylated goat anti-rabbit IgG (Sigma #B-8895). Dilute 1/1000 in 1% BSA/PBS.

4. ExtrAvidin conjugated Cy3 (Sigma #E-4142). Dilute 1/200 in 1% BSA/PBS.

5. Fluorescent Mounting Media (Dako #S3023)

6. Phosphate-buffered saline (PBS): 10 mM, pH 7.4, 150 mM NaCl.

7. Blocking Solution (10% BSA in PBS). Dissolve 1 g bovine serum albumin (BSA) (Sigma #A7888) in 10 ml PBS with gentle stirring.

8. 1% (w/v) BSA/PBS: Dissolve 1 g bovine serum albumin (BSA) (Sigma #A7888) in 100 ml PBS with gentle stirring. Use this solution to dilute antibodies.

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 to remove most of the liquid from tissues before each incubation. Avoid drying of specimens between steps. Use 50 - 100 µl or sufficient reagent to cover the sections for blocking or antibody incubations. Use 3-5 ml PBS each time when washing slides.

1. Place tissue in TissueTek O.C.T. and then freeze in liquid nitrogen. Store tissue at -80°C.

2. Cut 6-7 µm human liver O.C.T. sections, mount on gelatin-coated slides and fix in acetone for 20 min at room temperature. Place sections immediately into PBS.

3. Wash sections with PBS five times, shaking off excess PBS between each wash.

4. Incubate sections with blocking solution (10% BSA in PBS; 100 µl per section) for 60 minutes at room temperature in a sealed humid chamber.

5. Remove blocking solution and wash sections twice with PBS, shaking off excess PBS between each wash.

6. Carefully wipe around the sections to absorb excess moisture.
7. Add 100 µl Novozymes GroPep's IGFBP-3 Antibody for Immunohistochemistry (Catalog Code PAAJ1) (final antibody dilution 1/500 to 1/1,000 in 1% BSA/PBS). Incubate 1 hour at room temperature in a sealed humid chamber.

8. Wash sections with PBS five times, shaking off excess PBS between each wash.

9. Add 100 µl biotinylated goat anti-rabbit IgG (diluted (1/1000 in 1% BSA/PBS) to each slide. Incubate 1 hour at room temperature in a sealed humid chamber.

10. Wash sections in PBS five times, shaking off excess PBS between each wash.

11. Dilute ExtrAvidin conjugated Cy3 solution 1/200 in 1% BSA/PBS as required. Mix and briefly spin the diluted solution in an Eppendorf centrifuge. Pipette 100 µl of supernatant on each slide and incubate 40 min at room temperature in a sealed humid chamber. Keep sections out of light during and following this incubation.

12. Wash sections in PBS five times, shaking off excess PBS between each wash.

13. Cover each section with 1 drop of Fluorescent Mounting Solution. Add coverslip. Store slides at 4°C overnight to allow mounting medium to set before viewing sections in a UV microscope, using a green filter.


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