Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3100: Immunohistochemical (IHC) Detection of IGFBPs In Paraffin Sections Of Rat Embryonic Tissues Using Extravidin-Cy3 Staining

There are six classical Insulin-Like Growth Factor Binding Proteins (IGFBPs) that play an important role in stabilizing and regulating the activity of IGF-I and IGF-II in vivo.

This document details the ExtrAvidin-Cy3 staining protocol for the detection of IGFBPs 1-6, and IGFBP-rP1 in paraffin sections of rat embryonic tissues using Novozymes GroPep's IGFBP antibodies for immunohistochemistry. In this protocol, the paraffin-embedded tissue section is first de-paraffinized, re-hydrated, treated for Antigen retrieval, trypsinized, then incubated with the appropriate Novozymes GroPep IGFBP primary antibody, followed by a biotinylated anti-rabbit immunoglobulin (secondary antibody). The specifically bound secondary antibody is then visualized by ExtrAvidin conjugated Cy3.

All of Novozymes GroPep's IGFBP antibodies for immunohistochemistry (IHC) have been tested in rat embryonic tissues and anti-IGFBP-3 for IHC in human liver (OCT sections). Pre-absorption of the antibody with 1-2 µg (100 µl of 10-20 µg/ml) of the corresponding IGFBP for 1 hour at room temperature abolishes the immunohistochemical staining.

While the method described here was performed using rat tissue, it should also be suitable for human and mouse sections.

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. Snowcoat X-TRA slides (Surgipath #SP-00210)
5. Coverslips
6. Pipettes and other general laboratory equipment
7. UV microscope

Novozymes GroPep's IGFBP Antibodies for IHC and IGFBPs:

IGFBP-1 Antibody for Immunohistochemistry (Product Code PAAH1; 20 µg; final dilution 1/250).
IGFBP-2 Antibody for Immunohistochemistry (Product Code PAAI1; 20 µg; final dilution 1/500).
IGFBP-3 Antibody for Immunohistochemistry (Product Code PAAJ1; 20 µg; final dilution 1/500 to 1/1000).
IGFBP-4 Antibody for Immunohistochemistry (Product Code PAAG1; 20 µg; final dilution 1/250).
IGFBP-5 Antibody for Immunohistochemistry (Product Code PAAF1; 20 µg; final dilution 1/100).
IGFBP-6 Antibody for Immunohistochemistry (Product Code PAAE1; 20 µg; final dilution 1/500).
IGFBP-rP1 Antibody for Immunohistochemistry (Product Code PAAD1; 20 µg; final dilution 1/500).

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

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. Dissolve the appropriate Novozymes GroPep IGFBP Antibody for Immunohistochemistry (20 µg in 400 µl sterile distilled or Milli-Q water, a 1/20 dilution). Make up final dilutions (see details above) of the antibody 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. Biotinylated goat anti-rabbit IgG (Sigma #B-8895). Dilute 1/1000 in 1% BSA/PBS as required.

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

4. Sub-X solution to de-paraffinise sections (Surgipath P#03670).

5. Target Retrieval Solution (Dako #S1700)

6. Trypsin (Sigma #T-7409). Trypsin solution (0.625 g Trypsin / 250 ml PBS).

7. Fluorescent Mounting Media (Dako #S3023)

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

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

10. 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. Select the tissue sections desired for immunohistochemistry. Fetal rat sections can be used as a positive control. We routinely use 6 - 7 µm paraffin sections mounted on Snowcoat X-TRA slides.

2. Deparaffinize sections, 20 minutes in Sub-X solution (Surgipath, P#03670).

3. Re-hydrate sections with 100% ethanol for 2 minutes, then with 75%, 50%, 25% (all v/v) ethanol for 2 minutes each.

4. Soak sections in distilled or Milli-Q water for 2 minutes.

5. Antigen Retrieval:
Put slides into a Microwave proof ladle. Place ladle into a Microwave proof container and make sure all slides are covered with Target Retrieval Solution.

Microwave slides as follows:

i) Microwave 2 min on High (650W).

Top-up level of Target Retrieval Solution in slide container between each microwave step if necessary. Swirl solution in container to re-mix.

ii) Microwave 5 min on Medium-High, re-mixing and topping up with Target Retrieval Solution if necessary.

iii) Microwave 5 min on Medium.

6. IMPORTANT: Place microwave container in ice to allow tissue sections and Target Retrieval Solution to cool to at least 40°C before proceeding with Step 7.

7. Place slides into fresh Trypsin solution at 37°C for 3 min (0.625 g trypsin in 250 ml PBS), and then transfer slides into PBS solution to dilute the Trypsin solution.

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

9. Carefully wipe around the sections to absorb excess moisture.
10. Incubate sections with blocking solution (10% BSA in PBS; 100 µl per section) for 60 minutes at room temperature in a sealed humid chamber.

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

12. Dilute Novozymes GroPep's IGFBP Antibody for Immunohistochemistry in 1% BSA / PBS to give the required volume of the final dilution. Add 100 µl to each slide. Incubate 1 hour at room temperature in a sealed humid chamber.

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

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

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

16. Dilute sufficient 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.

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

18. Cover each section with 1 drop of Fluorescent Mounting Solution. Add coverslip. Store slides at -20°C overnight to allow mounting medium to set before viewing sections in a UV microscope, using a green filter.


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