Staining for Confocal Microscopy for Imaging of Proteins of Interest

Disclaimer. This SOP is subject to modification based on future insights and is not guaranteed to be universally applicable. It serves as a reference that can be adapted to specific experimental needs, acknowledging that procedures may vary between studies.

Purpose. Confocal microscopy stands as a pivotal technique in the realm of cell biology, allowing scientists to visualize and study the intricate details of cellular structures and proteins of interest with unprecedented clarity. Unlike traditional microscopy, confocal microscopy provides the ability to acquire in-focus images from selected depths, a feature particularly beneficial for thick specimens. This capability not only enhances the resolution and contrast of the images but also enables the visualization of moieties within the three-dimensional structure of cells. Such detailed imaging is crucial for understanding the complex interactions and functions within cells, thereby advancing our knowledge in various fields of biology and medicine.

Comprehensive. This SOP is designed for staining for confocal microscopy, tailored for use with a 4-well slide. This SOP facilitates the imaging of proteins of interest, though volumes may be adjusted for wells of different sizes.

Fixation:

Step 1: Carefully remove the medium using a pipette, avoiding the use of vacuum to prevent cell detachment.
Step 2: Wash with 100 μl of 1x PBS twice. Adjust the amount of PBS based on the well size.
Step 3: Add 200 μl of 4% paraformaldehyde (PFA) in 1x PBS under a chemical fume hood and incubate the chamber for 15-20 min.
Observation: At this point, you may pause the protocol and store the chamber at 4 °C, ensuring it is covered and kept flat.
Step 4: Under the fume hood, carefully remove the 4% PFA using a pipette.
Observation: It is now safe to continue work on your bench.

Blocking and Antibodies Incubation:

Step 5: Wash with 500 μl of 1x PBS twice.
Step 6: Add 200 μl of 0.1% Triton X-100 to permeabilize the cells, allowing for cytoskeletal staining and wait for 15 min. To prepare 100 ml of 0.1% Triton X-100, mix 99.9 ml of 1x PBS with 100 μl of Triton X-100.
Step 7: Wash with 500 μl of 1x PBS twice.
Step 8: Add 200 μl of 2% Bovine Serum Albumin (BSA) in 1x PBS to block non-specific binding sites and wait for 20 min. To prepare, mix 200 mg BSA with 10 ml of 1x PBS.
Step 9: Add 120 μl of the primary antibody diluted in 2% BSA in 1x PBS.
Step 10: Incubate the chamber at 4 °C overnight, ensuring it remains flat and covered.
Step 11: Retrieve and store the primary antibody.
Step 12: Wash with 500 μl of 1x PBS twice.
Observation: Perform these steps in a dark room to protect light-sensitive elements.
Step 13: Add 120 μl of the secondary antibody diluted in 2% BSA in 1x PBS and incubate the chamber at room temperature for 2 h.
Step 14: Wash with 500 μl of 1x PBS twice.

Cytoskeletal Visualization and Nuclear Staining with DAPI:

Step 15: Add 120 μl of Alexa Fluor 488 phalloidin (diluted 1:50 in 1x PBS) to stain the cytoskeleton. Cover the chamber and wait for 20 min. This step is crucial for visualizing the cytoskeletal structure. Alternative antibodies may be used.
Step 16: Wash with 500 μl of 1x PBS twice.
Step 17: Add 200/300 μl of DAPI (1 μM), cover the chamber, and incubate for 10-15 min to stain the nuclei.
Step 18: Wash with 500 μl of 1x PBS twice.
Step 19: Carefully remove the slide from the chamber. Apply a small drop of Mountant Permafluor to each well.
Step 20: Cover with a 22x50 mm glass coverslip, starting from one short side and gently moving to the other to avoid bubble formation.
Step 21: Allow to dry in the chemical hood for 30 min, covered with aluminum foil to protect from light.
Step 22: Store at 4 °C in the dark.

Figure. This published figure illustrates the expression of alpha-Smooth Muscle Actin (αSMA) in fibroblasts stimulated with Transforming Growth Factor-beta (TGF-β) following a 24-hour treatment with Dex-TPP/Mt labeled with FITC. In the image, αSMA is depicted in red, F-actin is shown in green, FITC-labeled Dex-TPP/Mt appears in yellow, and the nuclei stained with DAPI are in blue.

Profoundly. As Heraclitus said: “Physis Kryptesthai Phile (Nature loves to hide)!”. Modern techniques, such as confocal microscopy, allow us to reveal hidden mechanisms of life.

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Staining for Confocal Microscopy for Imaging of Proteins of Interest