The overall goal of this Laser Capture Microdissection protocol is to collect cells from specific brain regions effected by traumatic brain injury for molecular analysis. I'm Doctor Helen Hellmich. My graduate assistant, Harris Weisz, who is a student in the Human Pathophysiology and Translational Medicine program here at the University of Texas medical branch is going to demonstrate how we use laser capture microdissection methods in our brain injury studies.

This method could help to elucidate the mechanisms and consequences of TBI induced changes in discrete brain regions, such as the frontal cortex, which controls executive functions. And hippocampus which is important for memory dependent processes. The main advantage of this technique is that we can study the effect of a disease process on multiple discrete brain regions or individual cell types from the same brain.

Prior to any LCM procedure prior that includes transcriptional analysis, clean all surfaces with RNace eliminating detergent to mitigate the risk for contamination and RNA degradation. Retrieve the brain tissue from storage. And place in a cyrostat cooled to minus 20 degrees Celsius.

Allow the brain equilibrate to the temperature of the cryostat chamber for approximately ten minutes. Place the brain, ventral side up, on a gauze sheet on top of the cryostat stage. With a clean RNA razor blade cut off the posterior portion just rostral to the cerebellum and the portion just anterior to the optic chiasm.

Place a small amount of optimal cutting temperature medium into two separate cryo molds. Place the brain containing the frontal association cortex and nucleus accumbens core into one cryo mold interior side down. Add sufficient OCT medium to cover the tissue.

Repeat this step with the piece of brain tissue containing the hippocampus and suprachiasmatic nucleus. Allow the OCT medium to freeze completely for around 20 minutes in the cryostat. Once the tissue and OCT medium are completely frozen, squeeze a small amount of OTC onto a mounting head.

Press a frozen cryomold onto the mounting head and allow the OCT to freeze completely so that the mold containing the tissue is securely attached to the head. Secure the head attachment to the sectioning mount and tighten the screw. Adjust the cutting angle in relation to the cryostat blade with the adjustment levers to ensure the sections will be cut evenly.

Set the section thickness to 30 microns. When sectioning the tissue block containing the hippocampus and SEN, section until the optic chiasm is flattened at bregma 0.48 millimeters When the third ventricul becomes apparent. Then collect sections for the SEM from this point by gently placing room temperature polyethylene napthalate membrane slides on top of top of the sectioned tissue.

Visually ensure adherence to the slide by confirming that the tissue and OTC are completely melted onto the slide. Store all slides with tissue sections in the cryostat until staining. Stop collection sections at bregma 0.72 millimeters when the supraoptic decussation begins.

For hippocampus collection, section until the horns of granule cell layer dentate gyrus are visible at bregma 3.00 millimeters. Collect sections until the hippocampal CA sub fields are fused in the coronal sections at bregma 4.78 millimeters. This allows for complete collection of the hippocampus under the craniotomy and injury site.

Prior to staining, wash all dishware and the staining area in a chemical fume hood with RNase eliminating detergent. Take the rack of sides from the cryostat and place in the fume hood. Allow the slides to warm for 30 seconds.

Stain the sections with cresyl violet and clear with saline. Allow the rack of stained slides to air dry for no more than ten minutes at room temperature in the fume hood. Once dried, proceed immediately to LCM.

Prior to beginning any LCM procedure for RNA analysis, wipe down the collection area and the area around the device with RNAse eliminating detergent and 100%ethanol. Flip the power switch on the infared laser generating unit before turning on the microscope base and allow the system to initialize before the software is started from the desktop. Once the software is booted and run though it's initializing steps open the software set up panel and press the present stage button.

The modular stage will move into position where LCM macro caps and slides an be loaded and offloaded. Place up to three pen membrane slides into the holders with the frosted edge facing rightward. Select the cap and slide handling area next to the respective slide and click the MEM check box to tell the software that membrane glass slides have been loaded.

Then click on the desired slide to be captured first. To capture a tiled image for tissue location and orientation for cap placements, select image on the toolbar and select acquire overview. Place the cursor over the area on the tiled image, right click, and select place cap at region center.

The software will automatically place a cap over the selected position. After locating the infrared and UV lasers, choose the freehand drawing option in the select tools pane. Using touch screen stylus, draw around the perimeter of the region of interest while making sure not to lose contact between the touch screen and the stylus head.

Make sure to connect the beginning and end points together. For hippocampus collection, laser capture the CA1, CA2 and CA3 hippocampal sub fields starting at bregma 3.00 millimeters and using the completely formed granular layer of the dentate gyrus as a physical landmark of morphological identification. After collection move the LCM macro caps to the QC position and inspect for complete tissue adherence by visually ensuring the UV cut tissue is attached to the membrane.

It is imperative that the UV laser cuts around the entirety of the tissue. Failure to do so will result in incomplete tissue adherence to the LCM cap. Then quickly place each cap into an RNAse free 0.5 milliliter thinned walled reaction tube containing 100 microliters of cell lisus buffer.

Vortex the samples briefly to ensure complete lisus and store at minus 80 degrees Celsius until ready to perform downstream genomic analysis. The following are representative images of tissue sections collected from the epcilateral side of injury site with infrared and UV laser functions on the LCM system. This is an area of the frontal association cortex.

This image shows the same region after collection. And this is the tissue on cap after collection. This image shows the components of the CA1, CA2 and CA3 parameatal layers of the hippocampus located next to the fully formed horns of the granual layer of the dentate gyrus.

The CA2 and CA3 regions were collected and these cell populations are seen on the cap. Here, an area of the nucleus accumbens core proximal adrostral to the anterior commissure was targeted has has faithfully reproduced on the cap. Lastly, the suprachiasmatic nucleus rostral to the supraoptic chiasm was captured by LCM after TBI.

Once mastered, this technique can be used to process one brain in approximately eight hours. That includes tissue processing, staining and LCM. Following this procedure, other methods, like epigenetic analysis can be performed to answer questions regarding methylation patterns or micronic expression.

After watching this video you should have a good understanding on how to perform LCM on multiple brain regions on the same brain.

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