The overall goal of this procedure is to identify beta-amyloid plaques in brain sections from Alzheimer's disease mouse models prior to pre-embedding immunostaining and tissue processing for electron microscopy. This method can help answer the key questions in the field of neuroimmunology such as the interaction of microglial cell with the synapsis near amyloid beta plots. The main advantage of this technique is that it allows to prescreen brain tissue sections containing amyloid beta plots in the regions and layers of interest.
Though this method can be applied to Alzheimer's disease it can also be applied to any other diseases or tissues where amyloidosis is present. First, prepare a five milligrams per milliliter solution of methoxy X04 compound. Using a mirco balance, weight five milligrams of the methoxy X04.
Under a fume hood, dissolve the methoxy X04 in 100 microliters of DMSO and stir until a clear, greenish solution is obtained. Successively add 450 microliters of propylene glycol and 450 microliters of phosphate buffered saline while stirring. Keep the solution mixing at four degrees Celsius overnight.
The next day, a yellowish-green emulsion should be seen. After injecting methoxy X04, add a dose of 10 milligrams per kilogram of body weight and sacrificing the animal at the desired time point according to the approved methods, wash the para ormaldehyde fixed brain three times with chilled PBS. Then, using a sharp razor blade, remove the olfactory bulb and cut the brain coronally into two to three pieces of approximately equal height, all of which can be sectioned simultaneously in order to accelerate the procedure.
Glue the pieces of brain tissue onto the specimen plate secured into the tray. Make sure that the smooth cut surface sticks firmly to the specimen plate. Add PBS solution to the tray until the entire brain surface is completely submerged.
Place the tray in the vibratome and adjust the settings of the vibratome to yield 50 micron thick sections. Begin cutting, and if screening immediately transfer the sections into PBS using a fine paintbrush. Alternatively, sections can be placed in glass vials containing cryoprotected solution for storage at 20 degrees Celsius.
With the aid of a stereotaxic mouse brain atlas, select brain sections containing the region of interest and place each section into cryoprotectant in a well of a 24-well culture plate. Next, use a disposable pipette to add a droplet of PBS onto a microscope slide and then place the section on the droplet. Examine the section under a fluorescent microscope to identify regions containing methoxy X04 labeled a-beta plaques.
Without moving the microscope stage, capture images of the regions of interest in bright field as well as in fluorescence mode. Each image must show the same region in both fields to correlate the plaque to the structural region of the tissue section. Save and name the images taken according to the animal number.
Also record the well number in the plate and the field of the pictures taken. Place the section back into the designated well once the imaging is completed. Examine the next section in the sequence using the same procedure to avoid drying the sections.
To align the images, open the bright field and UV field images for the same ROI in ImageJ. Then, use the MosaicJ plugin to align the edges of the two images. Save the aligned and combined image according to the well number in a folder with the number of the particular animal.
Combine the images from different sections of the same animal to identify and localize the plaques in the region of interest. After the screening process is completed store the examined sections at 20 degrees Celsius is a 24-well culture plate containing cryoprotectant until immunostaining or further processing is carried out. First, prepare 1%osmium tetroxide solution in phosphate buffer in a glass vial.
As osmium tetroxide is photosensitive, cover the glass vial with aluminum foil to protect the solution from light. After washing in phosphate buffer, remove the buffer from the sections and spread them flat using a fine paintbrush. Be sure to flatten the sections immediately before adding osmium tetroxide as any folds in the sections will become permanent and attempting to flatten tissue post osmium fixation will only break the sections.
Use a transfer pipette to add osmium tetroxide to the sections one drop at a time until the section is immersed. Cover the well with aluminum foil to protect the sections from light and incubate for 30 minutes at room temperature. During this incubation prepare plastic resin in a disposable beaker.
Combine the components together in order and mix well using a 10 milliliter serological pipette until a uniform color is obtained. Transfer the prepared mixture to aluminum weighing dishes. These will receive the tissue sections once they have been dehydrated.
Once the incubation time has elapsed, dehydrate the sections in increasing concentrations of ethanol for two minutes each. Transfer the dehydrated sections from the 24-well culture plate into 20-milliliter glass vials. Then immerse the sections in propylene oxide three times for two minutes each time to remove residual ethanol.
Next, use a bent glass pipette tip or a fine paintbrush to transfer the sections from propylene oxide solution into the plastic resin and leave overnight for infiltration at room temperature. After infiltration, place the aluminum weighing dishes containing the specimens into a 50 to 60 degrees Celsius oven for 10 to 15 minutes. To embed sections on polychlorotrifluoroethylene film sheets first use a fine paintbrush to paint a thin layer of resin onto the section.
Then, move one section of tissue at a time from an aluminum weighing dish to the PCTFE film sheet. Remove excess resin from around the tissue, being careful not to disturb it. After moving all the sections from one weighing dish to the PCTFE film sheet, place a second PCTFE sheet over the first creating a sandwich of tissue and resin in between the two sheets.
Polymerize the resin in an incubator for three days at 55 to 60 degrees Celsius. The tissue is then ready for ultrathin sectioning and ultrastructural examination. The following images show dual imaging of one hippocampal section using bright field and fluorescence modes.
The regions and layers of interest are visualized under bright field. While the a-beta plaques are successfully localized using a UV filter at a range of 340 to 380 nanometers. This image shows a hippocampal section before pre-embedding immunostaining for IBA1.
A visible plaque is outlined by the dotted line. This image shows the same section after pre-embedding immunostaining for IBA1 and processing for transmission electron microscopy. Note that the plaque encircled by a dotted line is still visible upon tissue processing for electron microscopy.
Once mastered this protocol can be completed in one week if carried out properly. While attempting this procedure it is important to remember that every step must be done rigorously to reduce the probability of contamination and other structural degredation which will affect the quality of samples. Don't forget that working with osmium can be extremely hazardous and so you should always take precautions such as wearing a lab coat and gloves while performing this procedure.
Be sure to discard after using at the end of your experiment in accordance with your facility's guideline.
