Immunofluorescence Staining Protocol (Methanol permeabilization)
Licia Miller Product Manager
Immunofluorescence staining is a microscopic technique widely used in biomedical research. It detects and locates specific proteins at the cellular or tissue level by utilizing the binding of specific antibodies to target antigens and visualizing them with the help of fluorescent markers. This technique plays an indispensable role in many fields such as cell biology, pathology, and immunology due to its high sensitivity and specificity, providing researchers with a powerful tool to explore cell structure and function.
The basic principle of immunofluorescence staining is based on antigen-antibody reaction, in which a specific first antibody (primary antibody) recognizes and binds to the target antigen, and then a second antibody (secondary antibody) binds to the first antibody, and a fluorescent molecule is attached to the secondary antibody. This fluorescent labeling allows the location and distribution of the antigen to be visually observed through a fluorescence microscope, thereby achieving qualitative and quantitative analysis of specific proteins in cells.
This article aims to provide a standardized immunofluorescence staining experimental operation process through methanol permeabilization to ensure the reproducibility of the experiment and the accuracy of the results. By following these steps, researchers can effectively detect the expression and localization of target proteins and gain a deeper understanding of their role in biological processes. Correct experimental operation is essential for obtaining clear and reliable fluorescent images, and is also the cornerstone of the accuracy and reliability of scientific research data.
1. Fixation
Reagents Required
- 1× Phosphate Buffered Saline (PBS) , adjust pH to 8.0.
- Freshly prepared 4% formaldehyde, methanol-free.
Experimental Steps
NOTE: All subsequent incubations were performed at room temperature (20-25°C) unless otherwise stated.
1. Prepare the sample. Fixed frozen tissues can be directly used for the second step of immunostaining. If it is a cultured cell line or unfixed frozen tissue, it needs to be fixed immediately.
2. Cover the sample completely with 4% formaldehyde and fix it at room temperature for 15 minutes.
3. Rinse three times with 1× PBS, 5 minutes each time.
2. Permeabilization
Reagents Required
- 100% methanol , use ice-cold solution.
- Blocking Buffer: Purchase ready-to-use immunofluorescence blocking buffer (B751639) or prepare 1× PBS/5% normal serum/0.3% Triton X-100 buffer by adding 0.5 mL normal serum from the same species as the secondary antibody (e.g., normal goat serum) and 30 µL Triton X-100 to 9.5 mL 1× PBS. Store at 4°C.
Experimental Steps
1. Methanol Permeabilization: Cover the sample completely with ice-cold 100% methanol and incubate on ice or at 4°C for 10 minutes.
2. Rinse three times with 1× PBS , 5 minutes each time.
3. Block the specimen in blocking buffer for 60 minutes at room temperature.
3. Antibody Incubation and Imaging
All subsequent incubations should be carried out at room temperature unless otherwise noted requiring incubation in a humid light-tight box or covered dish/plate.
Reagents Required
-Antibody Dilution Buffer: Purchase ready-to-use immunofluorescence antibody dilution buffer or prepare 1× PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g BSA (B265994) and 30 µL Triton X-100 to 10 mL 1× PBS. Store at 4°C.
- Fluorescently conjugated secondary antibodies.
Experimental Steps
- For fluorescently labeled primary antibodies
1. Prepare the primary antibody using antibody dilution buffer (the recommended dilution concentration can be configured according to the product's official website).
2. Aspirate the blocking buffer , then add the diluted primary antibody and incubate overnight at 4°C.
3. Rinse three times with 1× PBS, 5 minutes each time.
4. Appropriate re-staining can be performed according to experimental needs.
5. Mount the sample for imaging.
For long-term storage, store samples at 4°C protected from light.
- For unconjugated primary antibodies
1. Prepare the primary antibody using antibody dilution buffer (the recommended dilution concentration can be configured according to the product's official website).
2. Aspirate the blocking buffer , then add the diluted primary antibody and incubate overnight at 4°C.
3. Rinse three times with 1× PBS, 5 minutes each time.
4. Dilute the fluorescein-conjugated secondary antibody with antibody dilution buffer and incubate the specimen in the dark for 1- 2 hours.
5. Rinse three times with 1× PBS, 5 minutes each time, and avoid light.
6. Appropriate re-staining can be performed according to experimental needs.
7. Mount the sample for imaging.
For long-term storage, store samples at 4°C protected from light.
For more product details, please visit Aladdin Scientific website.