NOTES: For both techniques the quality of proteins used for blocking or in dilution buffers is very important. The use of skim milk may cause problems, because milk may contain biotinylated proteinsw, that may affect the results. Use skim milk only, whe you are sure, that it does not contain biotinylated proteins.

BSA should be selected carefully. Some preparations may contain residual IGF´s and/or IGFBP`s. In our hands BSA from SIGMA, catalog  number A3059 gave good results. An alternative is fish gelatine from cold water fish, e.g. FLUKA catalog number 48717.

Conjugates: We have succesfully used the Streptavidin-Peroxidase Complex tablets from our western-ligand blotting kit.

Also the Vectastain ABC Reagents (Vector Labs, Burlinggame, CA, USA) has  been used succesfully

As substrates the IHC substrates for peroxidase from Zymed and KPL have been used sucessfully.

Avoid drying of specimens between steps. The use of humidifeid chambers is required.

Use 50 - 100 µl or sufficient reagent to cover the cells and sections completely.

1. Histochemistry on cells

Experiments have been made with biotinylated IGF-I. Concentrations of 1 to 10 µg/ml have been used. The other biotinylated IGF´s and analogues may require adjustment of the concentration. Also adjustment, depending on the number of available binding sites may be required.

1. Fixation: Fix cells in ice cold acetone/methanol (1:1 vol)  for one minute.
2. Block endogenous peroxidase with 1 % hydrogen peroxide in methanol at room temperature for 20 minutes.
   Alternatively a solution of  2 ml 30% (v/v) hydrogen peroxide solution and 0.1 g sodium azide to 100 ml of PBS with an incubation time of ten minutes may be used.
3. Wash cells three times with  phosphate buffered saline (PBS) and incubate cells with blocking solution (5 % BSA in PBS) for 20 minutes.
4. Wash sections twice with PBS with gentle shaking for 5 minutes.
5. Incubate cells with 1 % BSA in  phosphate buffered saline at a concentration of 1 to 10 µg/ml. Add sufficient solution to cover the cells completely. Incubate in a humidifed container for 90 minutes at room temperature.
6. Prepare a solution of 1 tablet Streptavidin-Peroxidase complex in 2 ml of 1 % BSA in TBS. The complete dissolution may require some minutes. The solution is stable for several days if stored refrigerated at 4 °C. Bring to room temperature before use.
7. Wash cells two times with PBS.
8. Add sufficient Strepatvidin-Peroxidase complex solution to cover the cells and incubate for 30 minutes at room temperature.
9. Wash cells two times with PBS.
10. Add a solution of 0.05 % (w/v) diaminobenzidine (DAB) in PBS and 0.1% (v/v) hydrogen peroxide.

The optimal DAB incubation time should be established for each kind of specimen. As a general guideline 2 minutes should be sufficient.  Check under microscope for positive brown colour staining. Avoid long DAB incubation time to minimise the background staining.

Rinse slides gently with tap water to wash off DAB solution, then rinse with PBS.

Lightly counterstain with Mayer's haematoxylin (Sigma, Catalog #MHS-1) for 1-5 minutes. The optimal concentration and staining time should be determined for different tissues and whether using fresh or old solution.

Wash cells briefly with 2 changes of tap water.

Dehydrate cells 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.

Mount coverslip using DPX mounting medium (BDH Chemicals).

Examine by light microscopy. This protocol should generate brown IGF-I staining with a pale blue counterstained background.

As a control for non-specific binding of Strepatvidin-peroxidase complex it is recommend to use cells, that are incubated without biotinylated ligand. As a control of specificity of ligand binding and competition experiments it is recommended to incubate the cells with the biotinylated ligand in the presence of a 25 fold to 50 fold excess of unlabelled ligand.

2. Protocol for tissue sections (may require modifications depending on the type of tissue section):

1. Select the tissue sections desired for immunohistochemistry.  3 - 6 µm paraffin sections mounted on gelatin-coated glass slides have been used succesfully.
2. Deparaffinise sections twice, 10 minutes each in 100% xylene (BDH Chemicals, Catalog #30575).
3. Hydrate sections with 100 % ethanol for 5 minutes, then with 80%, 50%, 30% (all v/v) ethanol for 5 minutes each.
4. Soak sections in distilled water for 5 minutes.
5. Incubate for 10 minutes in a solution of  2 ml 30% (v/v) hydrogen peroxide solution and 0.1 g sodium azide to 100 ml of PBS  to quench any endogenous peroxidase activity.
6. Wash sections with PBS with gentle shaking, three times for 5 minutes.
7. Carefully wipe around the sections to absorb excess moisture.
8. Incubate sections with blocking solution (5 % BSA in PBS) for 20 minutes.
9. Wash sections twice with PBS with gentle shaking for 5 minutes.
10. Incubate cells with 1 % BSA in  phosphate buffered saline at a concentration of 1 to 10 µg/ml. Add sufficient solution to cover the cells completely. Incubate in a humidifed container for 90 minutes at room temperature.
11. Prepare a solution of 1 tablet Streptavidin-Peroxidase complex in 2 ml of 1 % BSA in TBS. The complete dissolution may require some minutes. The solution is stable for several days if stored refrigerated at 4 °C. Bring to room temperature before use.
12. Wash sections with TBS with gentle shaking, three times for 5 minutes.
13. Add sufficient Strepatvidin-Peroxidase complex solution to cover the cells and incubate for 30 minutes at room temperature.
14. Wash cells two times with PBS.
15. Add a solution of 0.05 % (w/v) diaminobenzidine (DAB) in PBS and 0.1% (v/v) hydrogen peroxide.

The optimal DAB incubation time should be established for each kind of specimen. As a general guideline 2 minutes should be sufficient.  Check under microscope for positive brown colour staining. Avoid long DAB incubation time to minimise the background staining.

Rinse slides gently with tap water to wash off DAB solution, then rinse with PBS.

Lightly counterstain with Mayer's haematoxylin (Sigma, Catalog #MHS-1) for 1-5 minutes. The optimal concentration and staining time should be determined for different tissues and whether using fresh or old solution.

Wash sections briefly with 2 changes of tap water.

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.