The overall goal of this procedure is to isolate dopamine neurons from tissue sections on slides. This is accomplished by first sectioning frozen ventral mesencephalon tissue onto either stylized slides or pen membrane slides. Next dopamine neurons are labeled with a rapid fluorescent immunohistochemistry.
Then the tissue is dehydrated and loaded into the laser capture microscope. Then the labeled dopamine neurons are attached to the LCM cap using the infrared laser and the RNA or molecule of interest is isolated. The results demonstrate that RNA of sufficient quantity and quality can be obtained for quantitative PCR measurements using laser capture microscopy.
The main advantage of this technique over existing methods, such as gross dissection or micro punches, is that individual cell populations can be isolated or very discrete brain regions. To prepare for RNA isolation dip, either seline prep slides or polyethylene methylate or pen membrane glass slides into decontamination solution and use RNAs free water to wash them three times using a graded series of RNAs free ethanol dehydrate the slides and vacuum dry for 30 minutes with a cryostat cut tissue sections at 10 microns thick and mount on prepared RNAs. Free slides keep the samples frozen during sectioning to preserve RNA quality.
Next to carry out immunohistochemistry. Use a hydrophobic pen to outline tissue and allow them to dry then in a one-to-one acetone methanol solution. Fix the tissue at negative 20 degrees Celsius for 10 minutes After removing the tissue from the freezer, cover the sections with 100 to 200 microliters of tyrosine hydroxylase antibody NPBS and 1%tritton for 10 minutes.
Then use PBS with 1%tritton to rinse the slides before covering the sections with 100 to 200 microliters of goat anti rabbit IgG, labeled with Alexa Fluor 4 88 incubate for five minutes and then use PBS to rinse the slides twice. Next, dehydrate the samples integrated series of RNAs free ethanol for 30 seconds each as demonstrated earlier in this video. Incubate in xylene for one minute and a second, wash for five minutes.
Remove the slides immediately prior to use for LCM and allow them to air dry. After loading the LCM caps and the slides and setting up the LCM system according to the text protocol, adjust and aim the IR laser capture for strength. Place the cap in a region of the slide away from tissue on the capture laser toolbar.
Select enable, adjust the power pulse and number of 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. After adjusting the IR laser intensity, right click and select capture laser is here to aim the laser to perform LCM on individual dopamine neurons off of cline prep slides.
Begin by moving to the region of interest to capture the cells to capture individual cells off of a cline prep side. In the microdissection toolbar, select the LCM tab, then the single point icon. Next in the microscope toolbar, 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. Mark the cells of interest by selecting the single point icon and then clicking on the cells. Once the cells are marked on the microdissection toolbar, select the go cut and capture tab and the IR laser will automatically fire at all marked cells selected.
Move the cap away from the section and onto an open region of the slide to check if the cells are captured onto the LCM cap. When finished collecting tissue, move the cap by right clicking on the cap and selecting move to and then unload as outlined in the text protocol, use the slide processed for immunohistochemistry as a guide to select the region of interest from the tissue on the pen membrane. In the micro dissection toolbar, select the cut and capture tab, then select the cut line tab and using the immunohistochemistry labeled slide as a guide, outline the region of interest under the 10 x objective.
Test, fire and aim the IR and UV lasers as demonstrated earlier. In this video, in the microdissection toolbar, select the go capture and cut tab. Then move the LCM cap off of the tissue to determine if the tissue is removed from the section and attach to the cap.
When finished collecting tissue to remove the cap, right click on the cap and select move to then unload the micrographs in these figures demonstrate that LCM is capable of isolating individual tyrosine hydroxylase immunoreactive dopamine neurons using the IR laser or the IR and UV lasers because the most common use of cells in tissues isolated using LCM is RNA analysis. The procedures presented were optimized to preserve RNA integrity as seen by a relative reduction in height of the RRNA 28 S peak compared to the 18 S peak, RNA quality was reduced in LCM samples with lower integrity after immunohistochemistry, followed by acetone fixation compared to the whole brain RNA. To measure the quantity of RNA obtained from neurons acquired via L-C-M-R-N-A from dopamine neurons from the ventral teal area off of saline prep Slides were isolated based on the standard curve generated a total of 17.3, 24.8, and 50.9 picograms per microliter were isolated from 5, 100 and 200 dopamine neurons respectively.
E using QPCR to measure RNA quality, a range of concentrations of whole brain RNA and RNA from the dopamine neurons were reversed transcribed and the beta actin gene was measured from these measurements concentrations of three point 55, 6 0.82, and 20 point 58 picograms per microliter were calculated from 5, 100 and 200 dopamine neurons respectively. After watching this video, you should have a good understanding of how to use the laser capture microscope to isolate individual dopamine neurons or to isolate regions of dopamine neurons.
