IHC-P – Immunohistochemistry-Paraffin

Overview

Immunohistochemistry is an application used for demonstrating the presence and location of proteins in tissue samples. IHC-P does not produce as quantitatively intense data as Western blot or ELISA however it enables the observation of processes in intact tissue. This observation in tissue samples is made clear by the use of stained antibodies which then bind to specific proteins present in the tissue sample. This interaction is then visualized using either chromogenic detection or fluorescent detection.

Required materials

• Formaldehyde Fixative Solution

• Wash Buffer

• Ethanol

• Xylene

• Paraffin

• Incubation buffer

• Primary antibodies

• Secondary antibodies conjugated to HRP

• Cell and tissue staining reagents (DAB and hematoxylin)

• Antigen retrieval reagents

• Gelatin-coated slides

• Heat bath/pressure cooker

Protocol for fixing and sectioning paraffin-embedded Tissues

1. Fix tissue to preserve the morphology and antigenicity of the target molecule via vascular perfusion with the formaldehyde fixative solution. (If perfusion is not possible dissected tissue can be fixed via immersion in formaldehyde for 4-8 hours at room temperature.)

2. Paraffin is immiscible with water and the tissue must be dehydrated before adding the paraffin wax. Submerge tissue sample in ethanol for 30 min intervals until dry.

3. Imburse the tissue with paraffin wax at 58°C.

4. Cut tissue sections and float the sections in a 56°C water bath.

5. Mount these samples sections onto gelatin-coated slides.

6. Dry the slides overnight at room temperature. Slides can then be stored at 2-8°C or room temperature.

Deparaffinization

1. Wash the paraffin section with Xylene.

2. Wash sections with Xylene and ethanol.

3. Wash sections with ethanol.

4. Rinse with cold tap water.

5. Keep sections submerged in water. Drying of the section will allow the non-specific antibody to bind causing high background staining.

Antigen retrieval, Blocking, and Staining

1. Prepare your antigen retrieval buffer.

2. Submerge your tissue sections in a container with a retrieval buffer.

3. Prepare the heat bath or pressure cooker, depending on the antigen and buffer, parameters will need to be adapted and changed. Between the options of using a heat bath and a pressure cooker, the heat bath is a little slower.

4. Remove the container containing sections.

5. Rinse sections with PBS.

6. Block non-specific binding via incubation of sections with blocking buffer for 30 minutes at room temperature.

7. Incubate the sections with the primary antibody in dilute blocking buffer for 1 hour at room temperature or overnight at 4°C.

8. Wash sections with PBS to remove excess primary antibody.

9. Incubate sections with a detection system such as a secondary antibody HRP-conjugate and DAB substrate.

10. Use hematoxylin as a counterstain.

11. Dehydrate sections and then mount them with a mounting medium.

Results

Your results may consist of the images taken from a magnification range of 10X - 50X and a Histogram profile containing percentages from high positive pixels to low positive pixels and negative pixels.