The overall goal of this procedure is to more easily visualize fluorescent neurons within thick brain slices. This is accomplished by first embedding the brain in an aeros plug holder. The second step of the procedure is to section the brain into thick slices on the compressed tome.
The third step of the procedure is to optically clear the brain slices in a glycerol gradient. The final step of the procedure is to mount the cleared brain slices onto slides for fluorescent imaging. Ultimately, results can be obtained that show high resolution fluorescent protein reporter expression in neurons within thick brain slices, through epi fluorescent or confocal microscopy.
The main advantage of this technique over existing methods like thin section, serial fluorescent microscopy or multi photon imaging, is that it is much more rapid, less labor intensive and does not require expensive or sophisticated microscopy technique. To begin retrieve the mouse brain tissue that has undergone NC two fixation by cardiac perfusion and has been postfix for one to two hours at four degrees Celsius. Next, make a 2%high strength low gel point agro solution in PBS and heat in the microwave until dissolved.
Transfer the molten agros to a test tube and place an a 40 degree Celsius heat block to equilibrate the temperature while the agros equilibrates glue the fixed brain tissue onto the plunger of the specimen syringe. With Sano Aate glue submersed the mounted tissue in a saturated sucrose PBS solution to coat the brain surface and then draw down the plunger into the plunger housing. Pipette the warm aros into the plunger, completely covering the brain tissue while making sure not to introduce any air bubbles.
Finally, clamp the specimen syringe with a cold chilling block for about one minute. To set the aros, assemble the specimen syringe containing the embedded brain tissue into the compressed home. Align the edge of the blade closely with the outlet of the specimen syringe, and then fill the buffer tank with PBS.
Rotate the micrometer. Once slice thickness forward to extrude the embedded brain tissue out of the specimen syringe. To generate the first slice, press the start button on the control unit.
Repeat the process of advancing the embedded tissue and pressing the start button to generate the desired number of slices. Collect the slices with a transfer pipette and transfer them to a glass inhalation vial filled with PBS pipette off most of the PBS from the collected slices and replace with a 10%glycerol in PBS solution. Rock the vial gently at four degrees Celsius on a tabletop rotating shaker until the slices sink.
Continue the gradual clearing by repeating the decanting and refilling process with 25 50 and then 75%glycerol solutions. Pipette off the 75%glycerol solution and replace with 90%glycerol. Being careful not to introduce any bubbles and equilibrate at four degrees Celsius with gentle rocking.
Now that the slices have been maximally cleared, they will no longer sink, but will instead show neutral buoyancy. Cut double-sided adhesive into an open rectangle with a two to three millimeter border to fit the viewable area of a 25 by 75 millimeter microscope Slide using a pair of forceps. Arrange the brain slices within the border on the slide.
Gently lower the cover glass onto the adhesive tape. Being careful not to introduce air bubbles and apply pressure to create a seal aspirate any excess glycerol, and then seal the edges of the cover slip by applying clear nail polish. Once the seal has dried, the slide can be stored in a slide box at four degrees Celsius or imaged.
Immediately use using epi fluorescent microscopy. The expression pattern of a viral vector expressing enhanced GFP can be seen in a 200 micrometer thick coronal section of mouse brain tissue. Here, the highlighted area of the epi fluorescent image is shown at high resolution using confocal microscopy to reveal individual layer five cortical neurons.
After watching this video, you should have a good understanding of how to prepare fixed to thick brain slices for rapid and high resolution fluorescent imaging.
