This protocol allows resolving the membrane periodic skeleton in the axon initial segment of cultured neurons. By examining the localization of proteins insight can be gained into the function in the membrane periodic skeleton. The main advantage of this technique is its robustness and ease of use.
Structured illumination microscopy is capable of resolving the membrane periodic skeleton and sample preparation is easy and straightforward. Anyone with previous experience in fluorescence microscopy can readily implement this technique. This protocol is designed to maximize signal to noise ratio during sample preparation and imaging.
To begin, fix the neurons using 4%Paraformaldehyde for 12 minutes at room temperature. Wash the cover slip once in 0.2%BSA in phosphate buffer solution and incubate for 10 minutes in a 1%solution of Triton X in PBS at room temperature. Wash the cover slip with BSA in phosphate buffer solution, dilute the anti ankyrin G antibodies using BSA in a phosphate buffer solution at one to 200 ratio.
Add antibody solution to the cover slip and incubate overnight at four degrees Celsius. Wash the cells once in BSA phosphate buffer solution once in 0.1%Triton X in phosphate buffer solution and once in phosphate buffer solution. Dilute Fluoro 4 tagged, add more secondary antibodies in BSA phosphate buffer solution and add to the cover slip.
Wash the cells once in BSA phosphate buffer solution once in 0.1%Triton X in phosphate buffer solution, then once in phosphate buffer solution. Prepare a one micromolar solution of tagged Ferodin in phosphate buffer solution and add it to the cells. Wash the cells once in Bovine Serum Albumin phosphate buffer solution once in 0.1%Triton X in phosphate buffer solution, then once in phosphate buffer solution.
For mounting the cover slip on a glass slide, apply a drop of mounting media on a slide, dip the cover slip in deionized water, and tap it with a soft paper towel to remove excess water. Place the cover slip on the glass slide and incubate at room temperature for 24 hours. If possible, consult with the personnel from the microscopy facility before imaging and use an immersion oil calculator to select the immersion oil suitable for the sample.
Once the samples are ready, ensure the cover slips are clean and clear of any residue by cleaning them with a cotton tip dipped in water or ethanol. Place the sample in a 3D-SIM microscope and find the cell to capture the image. Adjust the power of the relevant laser lines.
And the exposure times to maximize the signal to noise ratio without significantly bleaching the sample. Set the upper and lower limits of the sample in the Z dimension and proceed to acquire a stack. Run the reconstruction algorithm on the stack to obtain a super resolved reconstruction.
Check the reconstructions for known artifacts and if necessary, adjust the parameters to correct them. Compare the SIM reconstruction to a wide field image to observe improvements in resolution. When performing multicolor structured illumination microscopy, use the alignment algorithm to align the different channels correctly, once a satisfactory reconstruction is ready.
Actin rings in the membrane periodic skeleton have a distinctive periodic appearance. Create a maximum intensity projection image in image analysis software using all the focal planes where the actin rings are visible. On the maximum intensity projection image, draw a perpendicular line across visible adjacent rings and record the fluorescence intensity along with the software's line plot profile function.
To calculate the mean inter peak distance, note the local maxima in the line profile and measure the distance between individual adjacent fluorescent intensity peaks. To evaluate the colocalization of different proteins with actin rings in the membrane periodic skeleton, run a colocalization analysis procedure on SIM reconstructions of actin and the candidate protein. Manually draw a selection to define the axon initial segment as a region of interest, for the easy colocalization plugin in the software platform, and run the analysis to calculate the Pearson's coefficient of correlation for colocalization.
During SIM image analysis, the reconstructions of image stacks, showed clear periodicity of actin rings. An anti ankyrin G antibody was used to visualize ankyrin G.The colocalization of ankyrin G and actin rings was tested in the axon initial segment using a colocalization analysis procedure to calculate Pearson's coefficient of correlation. The Pearson's coefficient of correlation for colocalization of ankyrin G and actin rings fluorescence was 0.36 plus or minus 0.03.
It is essential to use the right immersion oil and adjust the laser power and exposure time to make the grid lines visible. Once it is determined that protein is part of the membrane periodic skeleton, it's function can be investigated and established in maintaining the membrane periodic skeleton. For example, the effects of knocking down the protein on actin rings can be analyzed.
The development of super resolution microscopy techniques is the reason we know the axon initial segment contains membrane periodic skeleton comprising of actin rings, spectrum and ankyrin. SIM has enabled researchers to easily visualize membrane periodic skeleton components and look at actin rings in live cells.
