The lack of tunneling nanotubes specific markers restrict progress in the field and there is an increasing demand for a method to identify, characterize, and quantify tunneling nanotubes. Automatic detection methods are difficult to implement in without the expertise to develop an algorithm and/or change the existing algorithm, but our manual method is easy to implement with better precision. Long-range intercellular transfer via tunneling nanotubes is implemented in several ways in various disease models, including neurodegenerative pathology, viral spreading, and cancer.
Thus, studies on tunneling nanotubes to understand their role in pathogenesis are important. It is possible the tunneling nanotubes might break during the staining process. Avoid using cover slips and mounting on the slides, using glass bottom imaging dishes instead.
Modified fixation protocol helps to keep tunneling nanotubes intact. Demonstrating the procedure will be Deepak KV, a PhD student from the laboratory of Sangeeta NAF. To begin, wash the control and oligomeric A-beta treated cells twice with PBS for two minutes before fixation.
Prepare Karnovsky's fixative solution by using 2%formalin fixative and 2.5%glutaraldehyde dissolved in 0.1 molar phosphate buffer of pH 7.2. Then fix the cells in the imaging dish by adding Karnovsky's fixative solution for 45 minutes at room temperature. Prepare the incubation buffer by dissolving 0.1 gram of saponin in five milliliters of FBS and diluted by adding 95 milliliters of PBS.
Then wash the fixed cells twice using incubation buffer for two minutes. After fixation, add the first antibody against phospho-PAK1, add a dilution of one to 250 in the incubation buffer and incubate overnight at four degrees Celsius in a dark, moist chamber. The next day after 24 hours of incubation, wash the cells twice with the incubation buffer for two minutes.
Then add secondary antibody conjugated to Alexa Fluor 488 and Phalloidin 555. Incubate the cells in the dark, moist chamber for 1.5 hours at room temperature. After the incubation, wash the cells twice with incubation buffer for two minutes.
Stain the nucleus by adding DAPI in a one to 2000 dilution and incubate for five to 10 minutes at room temperature in the dark. Prepare DABCO mounting medium using 25 milligrams of DABCO in 90%glycerol and 10%PBS. To dissolve properly, adjust the pH to 8.6 using spectrophotometric grade dilute hydrochloric acid and keep the solution on a rocker for mixing.
As an anti-bleaching agent, add the DABCO mounting medium directly onto the imaging dish containing the cover slip at the bottom. Wait for at least one to two hours and directly proceed to perform confocal imaging. To identify tunneling nanotubes, capture Z-stack images of immunostained cells using a confocal laser-scanning microscope by first selecting the channels and clicking the required lasers sequentially in the windows track one, track two, and track three in the confocal software.
Click the option TPMT below the track one window to take the differential interference contrast images with fluorescence channels. Select the Acquisition tab of the software, click on the Z-stack tab, and wait for a window to open. Then click Live Scan to focus the cells at the bottom of the dish.
Select that focused image as the first stack, and then focus up to see the topmost part of the cell, and select that as the last stack. Stop live scanning and click the number next to the Optimal tab to fix the step size of the stacks which determines the number of slices and the intervals based on the thickness of the cells. Take sequential images of three channels of DAPI, FITC, and TRITC with 405 nanometer, 488 nanometer, and 561 nanometer lasers and capture with a pixel dwell time of 1.02 microsecond.
Capture images in the DIC channel with fluorescence channels to observe the cell boundary. Open the confocal images saved in czi data format in Fiji software for analysis. Select the Hyperstack option to see each Z-stack and channel of the image.
Scroll the channel and Z-stack scroll bars to select the exact stack of a particular channel of interest. Then select the F-actin stained channel first by scrolling the channel bar and manually scroll the Z-stacks to see each stack one by one. Identify the F-actin stained structures that seem to be connecting cells that are visible in the lower parts of the Z-stacks and are close to the surface of the imaging dish with Z equal to two as neurites.
Identify the tunneling nanotubes, the F-actin positive hovering cell to cell conduits by scrolling the Z-stacks toward the top from Z equals to four. Look for neurites near the lower parts of the Z-stacks toward the surface and observe that they start disappearing with the scrolling of Z-stacks toward the top at Z equal to six. Identify phospho-PAK1 positive tunneling nanotubes similarly as F-actin positive tunneling nanotubes by analyzing the hovering nature of the conduits from the Z-stacks.
As phospho-PAK1 staining is weaker than F-actin staining, look for phospho-PAK1 stained tunneling nanotubes at Z equal to four and at Z equal to six. Further, observe the DIC images to verify that the F-actin and phospho-PAK1 stained tunneling nanotube structures are membrane conduits between cells. In addition, merge F-actin and phospho-PAK1 channels to verify that the identified tunneling nanotubes are F-actin and phospho-PAK1 co-stained structures.
To quantify the tunneling nanotubes, count the total cell numbers and the identified tunneling nanotubes manually and represent the numbers as percentages. To distinguish F-actin and beta III tubulin positive tunneling nanotubes like hovering conduits from Z-stack images, merge F-actin and beta III tubulin channels, then analyze the Z-stacks of the merged images. Look for exclusively F-actin stained tunneling nanotubes that are faintly visible at Z equal to three and prominent at Z equal to six and Z equal to nine.
Similarly, identify F-actin and beta III tubulin. double-positive tunneling nanotubes like hovering conduits at Z equal to six and Z equal to nine. Identify other F-actin and beta III tubulin stained non-hovering protrusions from the lower parts of the Z-stacks.
Measure the diameter of the tunneling nanotubes using the Line tool in Fiji. Verify the scale of measurement by clicking Analyze and then set scale so that distance in pixels is set automatically from the czi images. Measure the diameters of the tunneling nanotubes at the XY plane.
Using the Volume Viewer plugin in Fiji which allows 3D re-slicing and threshold-enabled 3D visualization, split the Z-stack images into individual channels. Then crop the single channel Z-stack images to use 3D reconstruction view to highlight one or two tunneling nanotubes or neurites at a time. Pin a single tunneling nanotubes or neurite in the XY plane and mark XZ and YZ access cross sections.
Observe the neurites at the bottom of the XZ plane, in the XZ and YZ planes, and the tunneling nanotubes in the upper Z-stacks. Select the individual tunneling nanotubes or neurite to reconstruct the 3D volume view in the XZ plane. In the 3D reconstruction, observe the neurites at the bottom of the Z-plane and the tunneling nanotubes appearing as hovering structures connecting two cells without touching the bottom Z-plane.
Confocal Z-stack images of immunostained cells with F-actin and phospho-PAK1 were analyzed to identify tunneling nanotubes. Further, DIC images were analyzed to verify that the F-actin and phospho-PAK1 stained tunneling nanotubes structures were membrane conduits between cells. The cells were double immunostained with F-actin and beta III tubulin and the tunneling nanotube-like F-actin and the tunneling nanotube-like F-actin and beta III tubulin double positive membrane conduits were distinguished from only F-actin positive tunneling nanotubes.
The tunneling nanotubes co-expressed with phospho-PAK1 and F-actin were distinguished from other neurites'cell protrusions by constructing 3D volume view images based on their characteristic of hovering between two cells without touching the substratum. Usage of right fixative agent is important as imperfect fixation of the sample may lead to rapid proteolytic degradation of the target proteins and a reduction in the specific immunoreactivity.
