Immunofluorescence-Tissue

Immunofluorescence (IF) technology, based on the principle of antigen-antibody reaction, uses fluorescent antibody (antigen) as a probe to detect the corresponding antigen (antibody) in tissues or cells, and forms a fluorescent antigen-antibody complex in tissues or cells. To achieve the purpose of detecting, monitoring, analyzing and even purifying proteins.
Fluorescent probe types used for protein labeling include anthocyanins, Rhodamine, fluorescein, bioproteins such as GFP, and quantum dots. The fluorescent probe can be attached to the target protein by cyanine dye, Rhodamine dye, where fluorescein is labeled by attaching the cysteine, lysine, tyrosine or N-terminal of the target protein.
Depending on the results, it could eventually be possible to use techniques such as fluorescence microscopy to quantify and monitor target proteins, flow cytometry to sort proteins, or even fluorescent live cell imaging with multiple labeled proteins to monitor living cells^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Dewaxing

1.1 Dewaxing
1) Xylene I 10 min;
2) Xylene II 10 min;
3) Xylene III 10 min;
4) Anhydrous Ethanol I 3 min;
5) Anhydrous Ethanol II 3 min;
6) 95% Alcohol 3 min;
7) Rinse with running water 3 min.

2. Antigen Retrieval

The retrieval method can be adjusted according to the antigen, including boiling retrieval, high-pressure retrieval, and microwave retrieval.
1) Boiling Repair: Add approximately 1L of 1×Tris-EDTA (pH 9.0) buffer (adjust according to the number of sections) to a pressure cooker. Heat to a boil using an induction cooker for 3-5 minutes, ensuring no bubbles appear on low heat. Place the tissue sections into the pressure cooker, set the induction cooker to low power to maintain the temperature at 90-95℃, and cook for 20 minutes. Cover the cooker, but do not add a pressure valve (you can check during cooking to ensure the repair solution covers the sections and that there are no bubbles on the sections). Cool to room temperature for 10 minutes, then rinse with running water to cool to room temperature.
2) High-Pressure Repair: Add 1L of 1×citric acid (pH 6.0) buffer (adjust according to the number of sections) to a pressure cooker. Heat to a boil without tightly sealing the cooker. Place the tissue sections into the pressure cooker, add the pressure valve, and heat until uniform steam is released. Maintain this temperature for 2 minutes. Cool to room temperature for 10 minutes, then open the pressure cooker lid after the pressure valve has dropped. Rinse with running water to cool to room temperature.
Note: This method is suitable for antigen retrieval of difficult-to-detect or nuclear antigens.
3) Microwave retrieval: Add approximately 650 mL of 1×Tris-EDTA (pH 9.0) buffer (adjust according to the number of sections) to a transparent beaker, place it in a microwave oven, and heat on high until boiling; place the tissue sections in a retrieval box containing the boiled Tris-EDTA buffer (pH 9.0), and reduce the microwave oven to low to maintain a temperature of 95-100℃ for 20 min (divided into two 10-min sessions, checking in between whether the solution covers the sections and whether there are any air bubbles on the sections). After cooling to room temperature for 10 min, rinse with running tap water and cool to room temperature.

3. Permeabilization and Blocking

After slightly drying the sections, draw a circle around the tissue with a histochemical pen. Add a drop of a mixture of 0.25% Triton X-100 and 10% negative goat serum + 0.3M glycine (prepared with 1×TBS-T) to the circle to cover the tissue. Incubate the sections flat in a humidified chamber at room temperature for 30 min.

4. Primary Antibody Incubation

After removing most of the blocking solution with a suction aspirator, add a drop of primary antibody diluted with 1×TBS-T to the circle to cover the tissue (usually 100 μL/group). Incubate the sections flat in a humidified chamber at 4°C overnight (after removing from the 4°C freezer, allow to stand at room temperature for 30 min before proceeding with subsequent experiments).

5. Secondary Antibody Incubation

Wash slides three times with 1×TBS-T, 3 min each time. After patting the sections dry, add 100 μL of pre-diluted fluorescently labeled goat anti-mouse/rabbit IgG (containing DAPI 1:10000 or Hoechst 33258) diluted with 1×TBS-T to cover the tissue. Incubate at room temperature for 2-3 h.

6. Mounting

Wash slides three times with 1×TBS-T, 3 min each time. After washing, mount with anti-fluorescence quenching mounting medium (avoid air bubbles on the tissue during mounting to prevent interference with photography).

1. Regarding transparency, if the slice is relatively thin, it may not penetrate the film. But if you're studying proteins on the surface of the cell membrane, don't penetrate the membrane.

2. In the process of antigen repair, pay attention to prevent excessive evaporation of buffer when microwave oven heating, do not dry. The repair fluid and repair strength are determined according to the tissue.