IHC-Fr - Immunohistochemistry Frozen

Overview

Immunohistochemistry is an application used for demonstrating the presence and location of proteins in tissue samples. IHC-Fr 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

• Anhydrous ethanol
• PBS
• Antigen retrieval buffer-citric acid
• BSA
• Hydrogen peroxide
• HRP-conjugated goat anti-rabbit IgG
• DAB staining kit
• Hematoxylin staining solution
• Neutral gum
• Triton-X 100
• Tween-20
• DAPI
• Mounting Solution (IF)
• Heat bath/pressure cooker
• Oven

Protocol

1. Take frozen sections out from -80°C and dry slides in a 60°C oven for 30 minutes.
2. Treat with 0.5% Triton-X/PBS at 37°C for 15-20 minutes for permeabilization, then rinse the slides in PBS three times for 5 minutes each.
3. Perform antigen retrieval with citric acid (pH 6.0) by treating sections in heated citric acid at 90℃~95℃ for 15 min, then allow natural cooling to room temperature. Rinse the slides in PBS three times for 5 minutes each.
4. Block with 3% BSA at 37℃ for 1 hour.
5. Incubate with primary antibody overnight at 4℃, then rinse in PBS four times for 10 minutes each.
6. Incubate with 3% H2O2 at room temperature for 12 minutes, then rinse in PBS three times for 5 minutes each.
7. Incubate with HRP conjugated secondary antibody at 37℃ for 1 hour, then rinse with PBS three times for 10 minutes each.
8. Perform DAB staining at room temperature for 10-20 minutes and stop the reaction with distilled water.
9. Perform nuclear counterstaining with hematoxylin for 5-10 minutes.
10. Dehydrate sections through a series of 75%, 80%, 95%, 100% ethanol and 100%, 100% xylene.
11. Cover sections with neutral balsam.
12. Observe sections under a microscope.

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.