Laser capture MICRODISSECTION or LCM has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. Here LCM is used to dissect peripheral neurons from third instar oph larvae. Third instar larvae are embedded in a cryo mold and frozen then a cryo status used to cut serial tissue sections, which are placed on glass slides thawed and fixed in 70%ethanol.
The slides are then incubated with tripsin to remove residual larval cuticles and subjected to dehydration via ethanol gradient and xylene clearing. Next an LCM cap is placed on the tissue section and selective fluorescent neuron cells are captured using a pulsing laser. Finally, RNA is isolated from the captured cells for downstream applications such as quantitative R-T-P-C-R or microarray analyses.
Hi, I'm Dan Cox from the Department of Molecular and Microbiology and the Krasnow Institute for Advanced Study at George Mason University. Hi, I'm Ish AER from the laboratory of Dr.Dan Cox. Today we will show you a procedure for cell isolation and RNA purification of single or multiple drosophila peripheral neurons using laser capture microdissection.
We use this procedure in our laboratory to study the molecular mechanisms mediating class specific DIN rate morphogenesis. So let's get started. All of the procedures shown in this video are carried out in strictly RNAs free conditions following standard procedures.
Begin this protocol by collecting 30 to 40 aged match. Third instar larvae. Wash the larvae in double distilled water.
Rinse briefly in RNAs away, then wash one more time in double distilled water. To remove the RNAs away, using a clean Kim wipe completely wick off any excess water. Next, take a clean tissue embedding mold and add a thin layer of OCT.
Place the mold on a block of ice to cool to zero degrees Celsius. Using forceps, place the clean larvae and the cold OCT. The low temperature of the OCT will help reduce larval movement.
During the embedding process, arrange the larvae parallel to one another. Once the larvae have been arranged slowly fill the mold with OCT without disturbing them. Next snap.
Freeze the mold by placing it on a dry ice. This will ensure that the larva arranged along a single plane, which will maximize the number of cells available per section. Using a cryostat.
Cut frozen sections at five to eight micrometer thickness on plain labeled uncharged RNAs free glass microscope slides. Store the slides on dry ice, then transfer them to minus 80 degrees Celsius until ready for microdissection. All solutions for dehydration must be prepared fresh before each LCM session.
Complete dehydration of frozen larval tissue sections is essential for achieving optimal microdissection efficiency. Remove the slides containing the frozen larval tissue sections from the minus 80 degrees Celsius freezer and place them on dry ice. Remove a single slide from the slide box and immediately place it in a 50 milliliter conical tube filled with 70%ethanol for three to 10 seconds.
Then rinse the slide in a 50 milliliter conical tube filled with RNAs free double distilled water for five to 10 seconds to clear the cuticle from tissue sections which may interfere with the LCM capture. Gently pipette 50 microliters of 2.5%tryin directly onto the tissue section and incubate for five to 30 seconds at room temperature, rinse the sections briefly in a 50 milliliter conical tube containing RNAs, free double distilled water to remove the tripsin and any loosely adhere cuticle fragments. Complete the dehydration process by dipping the slides sequentially in the following solutions.
70%ethanol 95%ethanol, 100%ethanol, 100%ethanol, 100%xylene. And finally, one more time in 100%xylene. Following the dehydration, completely dry the slides under a mild airstream for 60 to 120 seconds at room temperature.
If possible, perform the microdissection in a humidity controlled room to prevent any reduction in efficiency due to increased humidity. First turn on the power for the microscope and laser control box. Load the caps your cap holder assembly with H-S-L-C-M caps.
Each cap is capable of capturing numerous cell bodies. Next, open the molecular devices software and enter the experiment details including slide number and cap lots number. Place the slide with the larval tissue sections on the microscope stage for microdissection.
Load a new HS LCM cap from the cartridge onto P cell two ELCM instrument and position it correctly with respect to the joystick to ensure proper positioning of the cap in relation to the capture area, locate the fluorescently labeled cells in the PPK GAL four U-A-S-M-C-D eight GFP or 21 7 G four UASM CD eight GFP transgenic report lines. The dendritic, arborization, or DA neurons of the peripheral nervous system can easily be identified by GFP fluorescence. To maximize specificity, choose cells with a robust fluorescent signal, a minimal cell overlap.
Next, adjust the power and duration laser pulse parameters to achieve a precise melted polymer spot whose size corresponds to the selected laser spot size for micro dissecting single class DA neurons. The following settings are recommended. Spot diameter 7.5 micrometers laser strength 30 to 50 milliwatts laser time, two to four milliseconds and one to two shots per cell.
Laser settings may vary depending on the tissue type tissue thickness and LCM cap type used. Ultimately, laser settings require empirical testing for optimal capture results. Next, subject the tissue to LCM.
During LCM, the cap is placed over the area of interest and a low power infrared laser. Pulse is directed through the cap onto a thin thermo lab polymer film above the cell or cells of interest. When the laser is pulsed, the film softens adheres to the target cells and res solidifies with the cells of interest attached to it.
When the cap is lifted from the tissue section, the cell or cells are microdisect from the surrounding tissue to capture more cells to a new field and repeat the process. Once the cells of interest have been captured, lift the LCM cap and place it on a clean slide to confirm the presence of the captured cells and absence of unwanted cells or debris. Image the HS LCM cap using fluorescence and transmitted light illumination.
Attach the cap containing the micro dissected cells to an extract your device. Then add 12 microliters of RNA extraction buffer to the cap surface and connect an inverted thin walled reaction tube as shown here. Place the assembly on an alignment tray and cover it with a preheated incubation block.
Then place the tray in a 42 degrees Celsius incubator for 30 minutes following the incubation centrifuge. The tubes containing the extracts at 800 times gravity for two minutes. After removing the tubes from the centrifuge, close them store the cell extracts minus 80 degrees Celsius until it is time to purify the RNA extract and column purify the RNA.
According to the pico pure RNA extraction kit instructions, DNA's treatment is optional and can be performed on column during the RNA purification as necessary. Once the RNA has been purified, store it at minus 80 degrees Celsius. Laser capture microdissection was used to isolate single class specific or multiple drosophila DA neurons from third instar larvae.
LCM allows for a high degree of precision and specificity in the isolation of drosophila D neuron cell bodies. Shown here is a representative image of a dehydrated and tripsin treated eight micron tissue section. Prior to performing LCM two class four DA neurons are labeled with GFP.
The arrowhead indicates the neuron that is highlighted for capture. The cell body of the highlighted neuron is cleanly micro dissected with high specificity from the tissue section. The insect shows an endon view of a single class four DA neuron captured on the LCM cap.
The total RNA purified from the isolated DA neurons was found to be of excellent quality as indicated by the presence of sharp 5.8 SAS and 28 s ribosomal. RNA peaks when analyzed on an Agilent 2100 bioanalyzer realtime quantitative reverse transcription PCR of the neuronal gene specific marker. Lev was performed to assess the neuronal specific enrichment of isolated cells using the delta delta CT method more than a 2.5 fold enrichment in the levels of lev was seen in the LCM captured DA neurons relative to the whole animal.
We've just shown you how to isolate and purify RNA from Joss or peripheral neurons via laser capture microdissection. When doing this procedure, it is important to optimize the trypsin concentration and the digestion time according to the tissue to be micros dissected under section thickness. Longer incubation times may result in loss of cells of interest from the tissue sections, whereas a short incubation time may be insufficient for removal of the larval cuticle resulting in inefficient LCM.
Importantly, all tissue dehydration solutions must be freshly prepared. Also to prevent damage to the LCM cap polymer xylene must be evaporated completely prior to LCM. Finally, to preserve RNA integrity from the purified cells, it's essential to maintain RNAs pre conditions throughout the procedure.
So that's it. Thanks for watching and good luck with your experiments.
