IF-cell Immunofluorescence Operation Video

Cellular Immunofluorescence (IF-cell) Operation Procedure

1. Relevant Reagent Formulations

2. Cell Fixation

1) When adherent cells have a normal morphology and reach a density of 50% to 70%, or suspension cells have a density of 1×10⁶, remove the cell culture medium.

2) Wash once with 1×PBS at room temperature.

3) Remove 1×PBS, add fixative 4% PFA, and fix at room temperature for 15 minutes. Alternatively, add ice-cold methanol and fix for 3-5 minutes.

4) Wash 3 times with 1×PBS at room temperature, 5 minutes each time.

3. Permeabilization and Blocking

When organic solvent fixatives such as methanol are used in the fixation step, permeabilization is not required. Note that excessive permeabilization can damage cell structure and cause signal loss, so the concentration and time should be strictly controlled.

1) Add permeabilization solution and incubate at room temperature for 15 minutes.

2) Remove the permeabilization solution, add 1×PBST, and wash twice at room temperature, 5 minutes each time.

3) Add sufficient blocking solution and incubate at room temperature for 1 hour.

4. Antibody Incubation

1) Dilute the primary antibody with antibody dilution buffer according to the instructions.

2) Remove the blocking solution, add diluted primary antibody to cover the cells, and incubate at 2-8°C overnight.

3) Remove the primary antibody, add 1×PBST, and wash 3 times on a horizontal shaker at room temperature, 5 minutes each time.

4) Remove PBST, add fluorescent secondary antibody, and incubate at room temperature in the dark for 45 minutes.

5) Remove the secondary antibody, add 1×PBST, and wash 3 times on a horizontal shaker at room temperature, 5 minutes each time.

6) Mount with DAPI-containing anti-fluorescence quenching agent and store in the dark.

Tissue Immunofluorescence (IF-tissue) Operation Procedure

Note：During the entire operation, the sections should be kept in a moist environment (wet box) to avoid drying, as this can easily cause non-specific staining.

1. Paraffin Section Dewaxing to Water

Before staining paraffin sections, dewaxing is necessary to ensure successful staining.

2. Procedure

1) Xylene I for 10 minutes;

2) Xylene II for 10 minutes;

3) Xylene III for 10 minutes;

4) Absolute ethanol I for 3 minutes;

5) Absolute ethanol II for 3 minutes;

6) 95% ethanol for 3 minutes;

7) Running water rinse for 3 minutes.

3. Paraffin Section Antigen Retrieval

The repair method can be adjusted according to the antigen, and permeabilization and blocking should be performed after repair.

1）Water Boiling Repair：Add an appropriate amount of 1×Tris-EDTA (pH9.0)/citric acid (PH6.0) buffer to a high-temperature container, heat it with an induction cooker until it boils for 3-5 minutes to ensure no bubbles under low heat; place the tissue sections in the container, adjust the induction cooker to low power to maintain the temperature at 90-95°C for 20 minutes, and cover with a lid. After cooling at room temperature for 10 minutes, rinse with running tap water to cool to room temperature. (For tissues that easily detach, such as bone, 60°C overnight repair can be used.)

2）High Pressure Repair：Add an appropriate amount of 1×citric acid (pH6.0) buffer to a pressure cooker, heat to boiling without a lid, place the tissue sections in the pressure cooker, add the valve, heat until uniform steam is emitted, maintain for 2 minutes, cool at room temperature for 10 minutes, then open the pressure cooker lid after the pressure valve drops, and rinse with running water to cool to room temperature.

*This method is suitable for antigen retrieval of difficult-to-detect or nuclear antigens.

3）Microwave Repair：Add an appropriate amount of 1×Tris-EDTA (PH9.0) buffer to a transparent beaker, place it in a microwave oven, heat on high power until boiling; place the tissue sections in it, adjust the microwave oven to low power to maintain the temperature at 95-100°C for 20 minutes (check if the solution covers the sections and if there are bubbles on the sections). After cooling at room temperature for 10 minutes, rinse with running tap water to cool to room temperature.

*Reason for Antigen Retrieval：During formaldehyde or paraformaldehyde fixation, some antigens in the tissue undergo protein cross-linking and aldehyde group blocking, resulting in loss of antigen activity. Through antigen retrieval, intracellular antigenic determinants can be re-exposed, improving the success rate of antigen detection.

4. Permeabilization and Blocking

After slightly drying the sections, draw a circle around the tissue with a histochemical pen, add blocking solution (10% goat serum + 0.3M glycine + 0.25% Triton X-100 prepared with 1×TBST) within the circle, and place in a wet box for permeabilization and blocking at room temperature for 30 minutes.

5. Primary Antibody Incubation

Aspirate the blocking solution, add primary antibody diluted with 1×TBST within the circle (usually 100 μL/group). Place the sections flat in a wet box and incubate at 4°C overnight (after taking out from the 4°C refrigerator, place at room temperature for 30 minutes before proceeding with subsequent experimental operations).

*When using directly labeled primary antibody for detection, mounting can be performed after this step.

6. Secondary Antibody Incubation

Wash the slides with 1×TBST 3 times, 3 minutes each time. After patting the sections dry, add 100 μL of pre-diluted fluorescent-labeled goat anti-mouse/rabbit IgG (containing DAPI 1:10000 or Hoechst 33258) within the circle to cover the tissue, and incubate at room temperature for 1-2 hours.

7. Mounting

Wash the slides with 1×TBST 3 times, 3 minutes each time. After washing, mount with anti-fluorescence quenching mounting medium (ensure no bubbles on the tissue during mounting to avoid affecting photography).

Frozen Section IHC-Fr Experimental Operation Procedure

1. Tissue Fixation

Perfusion Fixation：Through cardiac perfusion, first rapidly inject pre-cooled saline or PBS to rinse blood, then perfuse with sufficient pre-cooled 4% paraformaldehyde (PFA) fixative.

Sampling and Soaking Fixation：Quickly dissect the target tissue and soak it in 10-20 times volume of 4% PFA.

Fixation Conditions：Place in a 4°C refrigerator for overnight fixation (18 hours).

2. Sucrose Dehydration

Tissue Trimming：After fixation, briefly trim the tissue to the appropriate size and shape in PBS.

Gradient Dehydration：First transfer the tissue to at least 10 times volume of 15% sucrose solution (prepared with 1×PBS), dehydrate at 4°C until the tissue sinks to the bottom (about 8 hours or overnight). Then transfer to at least 10 times volume of 30% sucrose solution (prepared with 1×PBS), continue dehydrating at 4°C until the tissue sinks to the bottom (about 1-2 days).

3. OCT Embedding

Making Mold：Fold 2-3 layers of aluminum foil into an appropriate size square mold, ensuring no leakage. For tissues with directional requirements (such as brain), clearly mark A (Anterior), P (Posterior), and sample number on the mold.

Tissue Processing：Take the tissue out of the sucrose solution, gently and thoroughly blot the surface liquid with clean filter paper, ensuring all surfaces in contact with OCT are completely dry.

4. Sectioning

System Pre-equilibration：Start the frozen microtome, set both the cabinet temperature and sample head temperature to -20°C. This temperature is an optimized balance point that allows water-rich biological tissues and OCT to reach an ideal hardness that is neither too soft (sticky to the knife) nor too brittle (prone to fragmentation). Adjust to -15°C to -25°C according to actual conditions if necessary.

Component Precooling：Install a new or sharp blade and install the anti-roll plate. Allow it to fully equilibrate with the cabinet temperature to prevent section adhesion or abnormal curling due to temperature differences between components and the environment.

Tissue Block Fixation and Rewarming：Add a small amount of OCT as an 'adhesive' to the sample holder. Quickly adhere the frozen tissue block taken out from the mold to the sample holder, and place it on the microtome freezing stage to ensure it is firmly fixed.

Block Trimming and Sectioning：Adjust the knife holder so that the blade edge maintains a distance of about 1mm from the tissue block切面. Set the section thickness to 20-50 μm, quickly rotate the coarse trimming wheel until the largest and complete tissue切面 is trimmed out. Adjust the thickness to the target thickness (such as 10 μm) for formal sectioning. Rotate the wheel steadily and at a constant speed.

Mounting and Storage：Use adhesive slides to gently press the cut tissue sections to make them flatly adhere to the slides. Immediately mark clear and unique sample information on the slides. Store at -20°C for short-term (about 1 month); store at -80°C for long-term (at least 1 year) preservation of antigen activity.

5. Section Rewarming

Take the frozen sections out of the -80°C refrigerator, place them flat at room temperature, and rewarm for 30 minutes.

6. Antigen Retrieval

For most immunofluorescence experiments (especially rapid frozen sections or short-term paraformaldehyde-fixed samples), antigen retrieval is usually not required. If the antigen epitope is severely masked, select an appropriate retrieval method according to the primary antibody instructions or related literature. If not retrieving, proceed directly to the permeabilization and blocking step.

7. Permeabilization and Blocking

Draw Hydrophobic Circle：Use an immunohistochemical special oil pen to draw a circle around the tissue, keeping a distance of about 0.5 cm between the circle edge and the tissue edge to limit the liquid range and prevent sample drying.

Prepare Blocking Solution (50 mL Formula)：45 mL 1×TBS or 1×TBST 5 mL normal serum homologous to the secondary antibody 1.125 g glycine (about 0.3 M) 50–500 μL Triton X-100 (final concentration 0.1–1%, depending on antigen localization, higher concentration for nuclear localization targets) Mix thoroughly for use.

Blocking Incubation：Add blocking solution dropwise within the hydrophobic circle, ensuring complete coverage of the tissue. Place the sections horizontally in a wet box and incubate at room temperature for 30 minutes.

8. Primary Antibody Incubation

1) Gently shake off or aspirate the blocking solution (no washing required).

2) Add an appropriate amount (about 100 μL) of primary antibody working solution diluted according to the instructions, ensuring even coverage of the tissue.

3) Place the sections in a wet box and incubate at 4°C overnight (12–16 hours).

4) Take the wet box out the next day and rewarm at room temperature for 30 minutes.

5) Wash 3 times with 1×TBST buffer, 3 minutes each time.

9. Fluorescently Labeled Secondary Antibody Incubation

1) Pat dry the washing solution on the sections, keeping the tissue surface moist but not pooling.

2) Add an appropriate amount (about 100 μL) of fluorescently labeled secondary antibody working solution diluted as recommended.

3) Place the sections in a light-proof wet box and incubate at room temperature for 1–2 hours.

4) Wash 3 times with 1×TBST buffer, 3 minutes each time.

10. DAPI Staining/Mounting

1) After completing the last wash, gently aspirate excess liquid to avoid complete drying of the tissue. Mounting should be completed within 1–2 minutes.

2) Add a drop of DAPI-containing anti-fluorescence quenching mounting medium to the tissue. If the sample has been pre-stained with DAPI, use mounting medium without DAPI.

3) Use tweezers to hold one side of the coverslip and slowly cover from one end to avoid air bubbles.

4) For long-term storage, seal the edges of the coverslip with neutral resin or transparent nail polish to prevent evaporation of the mounting medium.

11. Photography

Objectively and accurately record the immunofluorescence results to provide reliable data for quantitative analysis.

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