Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

The overall goal of this procedure is to assess astrocyte morphology by analyzing three dimensional images of astrocytes that were acquired via confocal microscopy. This method can be used to evaluate morphological changes that can appear in various cell types, either in pathological conditions or as the effect of an intervention strategy. The main advantage of this technique is that many parameters associated with cellular architecture can be studied simultaneously.

Dr.Mariam Bodel will demonstrate this procedure Following the preparation of slides for immunohistochemical staining de parize sections by submersing them in xylene two times for 10 minutes. Incubate the slides for 10 minutes in each of 99.8%95%and 70%ethanol to rehydrate the sections after washing. PBS incubate the slides in a one to 1500 dilution of glial fibrillary acidic protein or GFAP primary antibody solution at four degrees Celsius overnight.

Following incubation, wash the sections in PB S3 times for five minutes. Then incubate the slides with a one to 100 alkaline phosphatase conjugated secondary antibody solution for one hour at room temperature. Again, wash the section in PB S3 times for five minutes, less than 30 minutes prior to use.

Add one drop of red chromogen solution to three milliliters of substrate buffer. Incubate each slide in 100 microliters of this solution for 15 to 20 minutes in the dark. Then wash the slides in PB S3 times for five minutes.

Finally, stain the nuclei with DPI diluted one to 500 in PBS. Apply one to two drops of Antifa medium on the section and immediately seal with a cover slip. Incubate the slides at four degrees Celsius for 24 to 48 hours before microscopy to ensure sealing.

To begin confocal microscopy, place a slide onto the microscope stage and select the 63 x objective. After opening the acquisition software, click on smart setup in the acquisition tab and select the proper floor of fours. Here, dappy and Alexa, floor 5 55 are used.

Next, click on best signal, followed by apply. Then click on set exposure to allow the computer to determine the optimal exposure parameters To optimize the image, open the channels window and switch the image to live. Adjust the focus if needed.

Then click on one au to optimize the pinhole, adjust gain for best intensity. Finally, set the digital gain between two and three. After this optimization, stop the live image and open the acquisition mode window.

Adjust the frame size by clicking on X by Y and select 10 24 by 10 24. Also choose a slow speed. Next, open the averaging tab and select a number greater than or equal to four.

Then click the snap button and save the captured 2D image. To set up 3D imaging, open the smart setup tab and mark the ZS stack item, which will cause the Zs stack window to open. To set the first and last positions for the Zack, again, click live and adjust the focus to the uppermost position of the astrocyte.

Click on set first. Now adjust the focus to the lowest position of the astrocyte, and click on set last, then stop the live view. Next, choose the interval by clicking on optimal.

To set the number of slices here, the interval is 1.01 micrometers. Finally, click on start, experiment, and save the images once the scan is complete. Also, acquire a 2D image using a 10 x or 20 x objective.

For fluorescence intensity measurements, select the rectangle or circle tool in the image window and highlight an area for measurement to quantify the volume and surface area of the astrocyte nucleus soma cell body and territory. Transfer the 3D images to an appropriate analysis software program, such as Velocity 6.1 in the analysis software. Create a new library and drag all of the images to the left gray column.

Select one 3D image and turn on one of the acquired channels of interest, such as dapi. For nuclear measurements, manually adjust the brightness to optimize the contrast. Now select the tools menu.

Choose make volume, and produce a single 3D image from the Zack images. Open the edit menu and click on properties. Ensure the X, Y, and Z properties are displayed.

Following this, choose the freehand ROI tool from the toolbar. Using the selected tool, draw a line around the DPI labeled nucleus to measure the volume and surface area of the astrocyte nucleus. Again, using the freehand ROI tool, measure the volume and surface area of the entire astrocyte by drawing a line around the soma following the surface of all the branches.

Click on the measurements menu at the top of the image window drag find objects to the top of the window and give a descriptive name to population one, select the associated color and click on measure, which will open a new window. In this new window. Choose either volume or surface area as the parameter and click okay.

To save the data, click on the measurement menu and select make measurement item. Select a new measurement item called from the window. Enter a file name for the data and click okay.

Finally, export the measurement data to an appropriate software package to perform statistical analysis and to generate data plots. Here, researchers compared astrocytes in fixed brain tissue from rats treated with amyloid beta one 40 or with amyloid beta one 40 and Ian. Treatment injection with the amyloid protein resulted in astrocytes with thicker and longer branches.

These astrocytes from Ian treated animals exhibited a stellate form with short and thin branches. Ian treated rats also demonstrated reduced astrocyte GFAP positive fluorescence in the brain slices. The morphometric analysis of specific astrocyte volumes revealed that the volume of the astrocyte nucleus cell body entire astrocyte and astrocyte territory were decreased with Ian treatment compared to amyloid treatment alone.

Once mastered this image, analyzes technique can be completed in a few minutes per cell if it's performed properly. While attempting this procedure, it's important to mark the structure with high precision. It is best to practice marking the structure of interest manually before starting the experiment after its development.

This technique paved the way for researchers in the field of cell biology, histology and pathology to explore a structure of a cell in a healthy or disease setting. After watching this video, you should have a good understanding of to measure 12 different parameters for a single cell using 3D pictures.