The overall goal of the following experiment is to visualize the expression and cellular localization of microglia macrophage phenotype markers following brain ischemia. First, cryo sections from ischemic mouse brains are stained to identify markers of microglia macrophage activation. The stained sections are subjected to three dimensional confocal imaging.
The resulting images are then processed to generate three dimensional renderings, and image analysis is performed to localize the expression of markers in clusters of cells. The resulting data demonstrate the effectiveness of three dimensional confocal analysis in properly assigning marker expression to a specific cellular body When two markers are expressed by the same cell, but in different subcellular compartments, colocalization alone may not be very informative. Using three dimensional analysis, as we will demonstrate here, allows for a better resolution and accuracy.
Though this method can provide insight into the complexity of brain response to ischemic injury, it can also be applied to other acute or chronic conditions where a dual role for microglia macrophages in injury and repair has been proposed. Or in those cases where signals need to be selectively localized in different special planes, Carlo Perigo and Stefan of Magali will take you through the different steps of the procedure. The following protocol utilizes mouse brain tissue in which ischemia was induced by permanent occlusion of the middle cerebral artery.
Use a cryostat to collect serial 20 micron coronal sections of the brain on gelatin. Covered slides bring all reagents and samples to room temperature before use. Please note the working dilution of antibodies and serum listed in their companion document resulted from trials made in order to obtain the best performance when different antibodies and serum are used.
Protocol needs to be validated. Begin by circling the sections on the slide using a liquid blocker pen to minimize the amount of reagents needed. Next, place the brain sections in a wet chamber using a one milliliter pipette.
Add room temperature PBS to the sections and incubate for five minutes to wash them. Then using a one milliliter disposable syringe with needle, remove the solution and repeat the wash once more After removing the second wash, proceed to stain the samples using a one milliliter pipette and a disposable syringe for all of the solution exchanges. The staining procedure should be performed as summarized.Here.
Please see the accompanying text for details, including washing steps. First, the cells are incubated with 1%hydrogen peroxide after blocking with normal goat serum. They're incubated with primary antibody against CD 11 B.Next, the slides are incubated with an appropriate secondary antibody followed by triss, NACL tween buffer, or TNT, then triss, H-C-L-N-A-C-L, blocking buffer, or TNB after the incubation with TNB.
The slides are incubated with streptavidin HRP followed by amplification diluent containing cyanide five tyrom, which will amplify the immunofluorescent signal. Next, the slides are incubated with NGS to block non-specific binding sites. Then they're incubated with a primary antibody against CD 68.
Following the incubation with the primary antibody, the cells are then incubated in the appropriate Fluor conjugated secondary antibody, followed by one microgram per milliliter of hooks to label the DNA. Once the staining is completed, mount cryo sections using prolonged gold when mounting sections, avoid generating air bubbles. Store the sections at four degrees Celsius in the dark until they're analyzed.
Acquisition of fluorescent signal should be performed within one week. This protocol utilizes an IX 81 microscope equipped with a confocal scan unit FV 500 with three laser lines argon krypton at 488 nanometers helium neon red at 646 nanometers and helium neon green at 532 nanometers plus a UV diode in the flu of U 500 software. Select the appropriate excitation lasers and dichroic mirrors.
Also set up image resolution at a minimum of 800 by 600 pixels. Next, place the slide on the stage of the microscope and using epi fluorescence, identify an area of interest, progressively increase the magnification to the 40 x objective. Then switch to laser scanning microscopy modality and run repetitive scans while adjusting the photomultiplier power level and the gain for each channel.
This will reduce signal overlap caused by contemporary excitation at different wavelengths. Keep the gain as low as possible to avoid unwanted nonspecific signals. Continuing the repetitive scan, adjust the focus and define the lower and upper extremes of the Z axis with total Z axis length of 10 microns.
Then stop the repetitive scan. Next, input the step size. It should be as close as possible to the pixel size, which is 0.225 microns.
This will give a standard one-to-one size ratio over the Z axis in the software Activate kalman filter at least two times. The kalman algorithm is an iterative function that eliminates random signal, such as single positive voxels belonging to background noise, thus improving signal to noise ratio. Now activate the sequential scanning mode to avoid bleed through effects.
Move to the midpoint of the Z axis and run an XY sequential scan. Check the setup of PMT and gain to ensure a satisfactory signal to noise ratio. Run XY, Z acquisition following the acquisition.
Export the data as a multi TIF file. Each multi TIF file typically contains three color channels and 44 focal planes for processing. Begin by opening imas software.
First, select the surpass view, then upload the multi TIF file. Next on the display adjustment panel, select the desired color for each channel Here. Red represents CD 11 B.Green represents CD 68, and blue represents the hooks DNA stain to remove noise from background, increase the minimum value on the display adjustment panel for each channel.
Then go to the section view to visualize a single XY focal plane with orthogonal projections of the x, Z and YZ axis. Move along the Z axis looking for colocalization, which appears as yellow pixels. Click on a yellow area to see whether the colocalization is present along the Z axis, which indicates that it belongs to a solid object Z axis.
Projections are visible in the right and bottom part of the figure. Take a snapshot, then go back to the surpass view. Next, in the edit dropdown menu, select crop 3D and outline a region of interest to isolate a cell or a cluster of cells.
Then on the display adjustment panel, select one channel. Select the surpass surfaces algorithm builder. Then to set up the algorithm, first, select a channel.
Then define threshold as the same minimum value as set in the display adjustment panel and define resizing if needed. Next, define smoothing as 0.200. Select the color panel and change the appearance as necessary.
Repeat the setup for the surpass surfaces algorithm builder for each channel. Then on the display adjustment panel, deselect fluorescence channels, and select all three surfaces. Finally, move the volume to find the best view and take a snapshot to determine the pattern of co-expression of MM markers.
The protocols for immunofluorescence and confocal acquisition described in this video were carried out in a mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery. A two dimensional view of acquired images shows that at 24 hours after ischemia, the lysosomal markers CD 68, which is shown in green, is expressed in the hypertrophic amid CD 11 B positive cells, which are seen in red present in the ischemic core. The Z axis projection shown in the bottom right demonstrates that the marker distribution documented in the single plane view is also present along the Z axis in the border zone.
The CD 11 B positive cells display hypertrophic cell bodies with processes highly positive for CD 68, suggesting that phagosomes are bound to the cell membrane. This indicates active PHA acidic behavior of microglial cells to assess co-expression of CD 68 and YM one, a marker of M two polarized. Mm.The fluorescent images underwent three dimensional rendering.
The three dimensional view of the acquired images shown here indicates the presence of CD 68 positive cells shown in green and YM one positive cells shown in red fluorescent voxels. Laying parallel, the observer's view may yield a co localized signal seen as yellow, which may not be related to the actual distance between markers. To define colocalization.
A single plane view with projections of the Z axis should be generated moving along the Z axis. Looking for colocalization. The Z axis projection is obtained as seen in the right and bottom part of this image.
Fluorescent voxels can be turned into solid objects by three dimensional rendering with assignment of marker expression to a specific cellular body. After a high magnification and 3D rendering, the two markers appear to belong to different cells that are in close proximity. After watching this video, you should have a good understanding of how to perform an immunofluorescence assay with multiple markers and dyes, and how to properly acquire and visualize three-dimensional images by confocal microscopy.
In addition, the post-processing analysis described that can be fruitfully exploited to analyze any co-expression or colocalization at cellular level, or in those cases where signals need to be selectively localized in different spatial planes.
