Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3300: Immunohistochemical (IHC) Detection of TGF-ß2

Introduction

Transforming Growth Factor-ß2 (TGF-ß2) is one of a family of transforming growth factors identified in mammals. TGFßs mediate their activity by high affinity binding to the TGFß type II receptor which signals through the serine/threonine kinase pathway. Immunohistochemical detection of TGF-ß2 is one of the important tools for studying the local expression of TGF-ß2 peptide in tissues. This document details a Streptavidin-Cy3 protocol for TGF-ß2 detection in TissueTek O.C.T. frozen tissue sections using Novozymes GroPep's TGF-ß2 Monoclonal Antibody. (Catalog Code MAB). In this protocol, the frozen sections are incubated first with this monoclonal antibody (primary antibody), then with a biotinylated anti-mouse IgG antibody (secondary antibody). The specifically bound secondary antibody is then visualised with the fluorescent streptavidin-Cy3.

Novozymes GroPep's TGF-ß2 Monoclonal Antibody. (Catalog Code MAB1) has been used for human tissues.

Novozymes GroPep Reagents

Store lyophilized reagents at 2-8°C.

Primary Antibody: Novozymes GroPep TGF-ß2 Monoclonal Antibody (500 µg)
Dissolve in 1 ml of sterile distilled or Milli-Q water. Aliquot and freeze at -20°C. Dilute in phosphate buffered saline pH 7.4.

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; Cat.No. 4583)

2. Cy3 Streptavidin (Sigma Chemical Co., St Louis, MO, USA; Cat. No S6042)

3. Secondary Antibody: Biotinylated sheep anti-mouse IgG (Sigma Chemical Co., St Louis, MO, USA; Cat. No B9140)

4. Shandon Immuno-Mount (Life Sciences Intl, Cheshire, UK; Available through Jomar Diagnostics, Adelaide, Australia; Cat. No. 9990402)

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


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 -80oC. 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 1/10 to 1/50 in PBS for human tissues. Incubate in a humidified chamber for 60 min at room temperature.

4. Rinse sections 3-4 times in PBS.

5. Add 100 µl per section of the secondary antibody, diluted 1/200 in PBS. Incubate at room temperature for 60 min.

6. Rinse sections 3-4 times in PBS.

7. Add 100 µl per section of Cy3 streptavidin, diluted 1/300 in PBS. Incubate at room temperature for 40 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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