Laser Capture Microdissection – A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

NOTE: Brain tissue from mice was used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and study protocols were approved by the Institutional Animal Care and Use Committee at Mississippi State University.

1. Preparation of Slides and Tissue

1. Use either Silane-prep slides or polyethylene napthalate (PEN) membrane glass slides.
2. For RNA isolation, dip slides in RNase decontamination solution, wash 3 times with RNase-free water then dehydrate with a graded series of RNase-free ethanol and vacuum dry for 30 min.
3. Cut tissue sections at 10 µm thickness using a cryostat and mount on the prepared RNase free slides. Keep the sections frozen during sectioning to preserve RNA quality.
   NOTE: Be sure that the tissue is approximately 4 mm away from the edges of the slide as the LCM cannot remove tissue that is too close to the edges of the slide. In order to remove tissue from a PEN membrane slide, ensure that the tissue is 4 mm from the left, top and bottom and 7 mm from the right side of the slide which are the dimensions of the membrane that is not attached to the slide.
4. For isolation of individual cell populations using direct immunohistochemistry, section tissue onto either the Silane-prep slides or PEN membrane slides. For isolation of regions of tissue using indirect immunohistochemistry guided techniques, mount one set of sections on a Silane-prep slide for immunohistochemistry and alternate sections on either a PEN membrane slide and/or a Silane-prep slide to be used for LCM.

2. Tissue Preparation for Laser Capture – Rapid Tyrosine Hydroxylase Immunohistochemistry for Direct Laser Capture of Immunoreactive Cells

1. Outline tissue with a hydrophobic pen and allow to dry.
2. Fix tissue in acetone-methanol (1:1) solution at -20 °C for 10 min.
   NOTE: Our experience has shown that the acetone-methanol fixation resulted in much more consistent immunohistochemistry than acetone or methanol alone.
3. Rinse slide in phosphate buffered saline (PBS) with 1% Triton (RNase free).
4. Cover sections with 100-200 µl PBS with 1% Triton with tyrosine hydroxylase antibody diluted 1:100 with 400 U/ml RNasin. Incubate for 5-10 min.
5. Rinse briefly in PBS twice and PBS-1% Triton.
6. Cover tissue with 100-200 µl of goat anti-Rabbit IgG labeled with Alexa Fluor 488 diluted 1:100 in PBS-1% Triton with 400 U/ml RNasin and 50 ng/ml DAPI. Incubate for 5 min.
7. Rinse 2 times in PBS then dehydrate 30 s in a graded series of RNase free ethanol (75%-75%-95%-95%-100%-100%).
8. Incubate in two washes of Xylene for 1 min then 5 min.
9. Remove slides from Xylene immediately prior to use for LCM and allow to air dry.

3. Tissue Preparation for Laser Capture – Tyrosine Hydroxylase Immunohistochemistry for Indirect Laser Capture of Immunoreactive Regions

1. Process one slide with sections adjacent to sections mounted on Silane-prep and/or PEN membrane slides for immunohistochemistry.
   NOTE: Procedures used here are similar to those previously reported ⁵. For the demonstration images, the diaminobenzidine (DAB) chromagen reaction was used to visualize the tyrosine hydroxylase neurons of interest.
2. For slides used for LCM, fix for 5 min in 100% acetone at 4 °C.
3. Dehydrate tissue in a graded series of RNase Free ethanol (75%-75%-95%-95%-100%-100%) for 1 min each.
4. Incubate in two washes of Xylene for 1 min then 5 min.
5. Remove slides from Xylene immediately prior to use for LCM and allow to air dry.

4. Using the Laser Capture Microdissection System

NOTE: The LCM application program has 10 Tool bars and 2 windows to control the microdissection procedure. Tool Bars: 1. Microscope, 2. Capture Laser, 3. Cutting Laser, 4. Study, 5. Navigation, 6. Materials, 7. Capture Groups, 8. Microdissection, 9. Font, 10. Annotation. Windows: 1. Live Video and 2. Road Map.

1. Open the door by selecting the “Open door” tab in the Materials tool bar in LCM system software.
2. Load slides on the three available slots. For this demonstration, load one slide with tyrosine hydroxylase immunohistochemistry visualized with DAB, one Silane-prep slide and one PEN membrane slide each labeled for rapid fluorescent tyrosine hydroxylase immunohistochemistry. In a typical experiment, use either unlabeled acetone fixed sections with a reference slide labeled for tyrosine hydroxylase using DAB or slides directly labeled with fluorescent tyrosine hydroxylase for laser capture.
3. Load the LCM caps into the available slot.
   NOTE: Macro and HS LCM caps are available for use. The type of LCM cap to use depends on the number of cells or amount of tissue to be collected. Macro slides are listed to collect up to several thousand cells while HS caps can collect several hundred. The Macro slides allow for more tissue collection but require 50 µl of lysis buffer. The HS cap, when used with the adaptors, requires only 10 µl of lysis buffer which allows for greater proportional recovery of RNA.
4. Close the door.
5. Load the slide to activate the Road Map window which provides a working view of the entire slide, this allows for selection of the region of interest (Figure 3). View the Road Map on Live video window to navigate to the area of interest.
6. In the Materials tool bar, identify the PEN membrane slides by checking the box above the slide slot. To supply cap properties, right click on the caps in the Materials tools bar and select “Cap supply properties”. Check the boxes indicating the position of the caps and select either Macro or HS caps.
7. Select the “Fluorescence” tab in the Microscope tool bar to turn on the fluorescent lamp and allow the fluorescent lamp to warm up. Use the Blue and UV filters to visualize the Alex Fluor 488 and DAPI, respectively. Use the “Adjust Camera” tab to increase the sensitivity of the image in order to see the fluorescently labeled cells.
8. In the Materials tool bar, right click on a cap and move it to the slide of interest. Double click on a location in the Road Map to select the region that appears in the Live Video window. Move the cap around on the slide by selecting an area on the Road Map (double left click) and then right clicking and selecting “Place cap at center region.”
9. Before using the IR capture laser, aim and adjust it for strength. Place the cap in a region of the slide away from tissue. On the Capture Laser tool bar, select “Enable.” Adjust the Power (3-100 mW), Pulse (100-1000000 µsec) and # Hits of the laser. Fire the IR laser when the cap is over a portion of the slide without tissue and check to see if the laser sufficiently melts the LCM cap membrane.
   NOTE: This is called wetting which is melting the polymer membrane on the cap so that it contacts the glass slide. Sufficient wetting is visible as a dark ring fused to the slide with the center of the ring clear (See Figure 4 for examples of sufficient and insufficient melting). If wetting is not sufficient, adjust the power and pulse, and test fire again until this is achieved. Additionally, multiple fires of the laser can also be used.
10. When the IR laser intensity is adjusted, right click and select “Capture laser is here ” to aim the laser.
    NOTE: This step tells the computer where the IR capture laser is aimed at the tissue. This step of aiming the laser is critical and needs to be repeated every time the cap is moved or the objective is changed.

5. Laser Capture Microdissection of Individual Dopamine Neurons off of Silane-prep Slides

1. Move to the region of interest to capture cells.
   NOTE: The example of isolating dopamine neurons is shown here.
2. To capture individual cells off of a Silane-prep slide, select the “LCM” tab then the “single point” icon in the Microdissection tool bar.
3. In the Microscope tool bar, use the “Shutter” tab to switch between fluorescence and bright field with fluorescent filters tabs to choose the appropriate filters and use the objectives tabs to change the magnification. Once the cells of interest are visible in the Live video window, adjust the spot size used to mark cells of interest to the approximate size of the cells. Do this by clicking the Spot tab on the Capture Laser tool bar then clicking on one side of a stained cell, followed by clicking on the opposite side.
   NOTE: This calculates the diameter of the spot and displays the value.
4. Mark the cells of interest by clicking on them while the “Single point” icon is selected. Do this to place a spot marker on each cell. Here, the Blue filter and the 10X objective are used to visualize the dopamine neurons. Retest and aim the IR laser on the cap where there is no tissue beneath it as described in steps 4.9 and 4.10.
5. Once the cells are marked, select the “Go – cut and capture” tab on the Microdissection tool bar and the IR laser will automatically fire at all marked cells selected. Additionally, if needed, fire the IR laser manually at any point of interest (Figure 5D).
6. Move the cap away from the section and onto an open region of the slide to check if the cells were captured onto the LCM cap. Ensure that the cells of interest are removed from the slide (Figure 5B, E) and attached to the LCM cap (Figure 5C, F). Document this process by clicking on the “Capture image” tab in Microscope tool bar.
7. When finished collecting tissue, remove the cap by right clicking on the cap and select “Move to” then “Unload.”

6. Laser Capture of Individual Dopamine Neurons off of PEN Membrane Slides

1. To capture individual cells off of PEN membrane slides using both the IR and UV lasers, select “Cut and Capture” tab in the Microdissection tool bar. Select the “single point” tab to mark the cells of interest. Use the “Cut line” tab to outline each cell. Be sure to test the IR laser as described in step 4.9 and 4.10 and test the UV laser to determine the minimum intensity necessary to cut thought the PEN membrane and tissue (Figure 4 and 5)
2. Select the “Capture and Cut” tab on the Microdissection tool bar. When collecting individual cells using this technique, be sure to fire the IR laser first followed by the UV laser as these small pieces can be lost if they are cut out prior to being affixed to the LCM cap.
   NOTE: This process can also be done manually by firing the IR laser at a point of interest and the cells individually outlined with the UV laser.
3. After collecting the cells of interest, move the LCM cap as described above in step 5.7 to determine if the cells were removed and attached to the cap (Figure 6).
4. When finished collecting tissue, remove the cap by right clicking on the cap and selecting “Move to” then “Unload.”

7. Capturing the Ventral Tegmental Area from Silane-prep Slides

1. To identify the region of interest, use the slide processed for immunohistochemistry.
   NOTE: For this demonstration the ventral tegmental area is isolated (Figure 7A).
2. Load a LCM cap and test the lasers as in steps 4.9 and 4.10.
   NOTE: Typically, a Macro LCM cap is used but an HS cap can also be used (See Figure 8).
3. To isolate a region of tissue (i.e., the ventral tegmental area) from the Silane-prep slide, select the “LCM” tab in the Microdissection tool bar, then select the “polygon-exterior” tab. Outline the region of interest in the Live Video window. Depending of the size of the area for collection, outline the region of interest at low magnification (2X Objective). For collection, however, the instrument uses the 10X objective so adjust and aim the IR laser under the 10X objective prior to capturing.
4. Select the “Go – capture and cut” tab in the Microdissection tool bar.
   NOTE: The computer will automatically fire the IR laser throughout the entire selected area (Figure 7F,I). If the LCM membrane has not been melted sufficiently throughout the area, the laser can be fired manually or the entire process repeated.
5. Move the LCM cap off of the tissue to determine how well the tissue was collected.
   NOTE: One pass using this method typically leaves some tissue behind on the slide (See Figure7G). If this occurs, repeat the selection and capture step above. Repeating this step can increase the amount of tissue collected (Figure 7J).
6. When finished collecting tissue, unload the cap by right clicking on the cap and selecting “Move to” then “Unload.”

8. Capturing the Ventral Tegmental Area from PEN Membrane Slides

1. Use the slide processed for immunohistochemistry as a guide to select the region of interest from the tissue on the PEN membrane slide as above.
2. Select the “Cut and Capture” tab in the Microdissection tool bar. Select the “Cut Line” tab and outline the region of interest using the immunohistochemistry labeled slide as a guide.
   NOTE: The program will automatically add spots for the IR laser to attach the tissue to the cap. Use the “single point” tab to add additional spots to better secure the tissue to the cap.
3. Under the 10X objective, test fire and aim the IR and UV laser as described in 4.9 and 4.10. Test that the UV laser can penetrate the membrane on the slide and that this cut corresponds to the location on the computer screen. Use a UV laser strength that is just sufficient to cut the membrane and tissue but does not damage the tissue (Figure 5).
4. Select the “Go – capture and cut” tab in the Microdissection tool bar.
   NOTE: The computer will automatically fire the IR laser at all labeled spots within the tissue to attach it to the cap then fire the UV laser to cut the slide PEN membrane and tissue (Figure 7C). Additional IR melted spots may be necessary to adequately attach the tissue to the LCM cap, which can also be added manually.
5. Move the LCM cap off of the tissue to determine if the tissue was removed from the section (Figure 7C) and attached to the cap (Figure 7C).
6. When finished collecting tissue, to remove the cap, right click on the cap and select “Move to” then “Unload.”

9. Isolation of RNA

1. To collect RNA from the captured cells or tissue, incubate the LCM cap membrane in RNA lysis buffer for 30 min at 37 °C.
   NOTE: For HS caps, an adaptor is available that allows for the use of only 10 µl of lysis buffer on the cap and attaches to a 0.5 ml microcentrifuge tube. The Macro caps require 50 µl of lysis buffer and attaches directly to a 0.5 ml microcentrifuge tube.
2. After incubation, spin the lysis buffer down into the 0.5 ml microcentrifuge tube.
3. To ensure complete lysis of all the cells or tissue, peel off the LCM cap membrane of the cap and place in the lysis buffer.
4. To test for RNA integrity, use RNA isolated from the tissue remaining on the slide after LCM to measure RNA integrity.
   NOTE: Due to the limited sample RNA obtained with LCM, it is typically not practical to test RNA integrity on LCM isolated RNA, however, RNA from the remaining tissue provides a good indicator of any RNA degradation due to fixation, immunohistochemistry and time during the LCM procedure.