1,093
edits
Changes
Added operating procedure
Subsequent to X-Gal staining, the sections were washed in DEPC-dH2O for 5 min and dehydrated by the following procedure: 70% ethanol for 30 s, 95% ethanol for 30 s, 100% ethanol for 30 s, xylene for 5 min, air dried in a fume hood for 15 min, and then desiccated with Drierite (W.A. Hammond Drierite, Xenia, OH) for 1 h. X-Gal stained cells in the CeA, cortex and hippocampus were microdissected using the PixCell II LCM system (Arcturus, Mountain view, CA). A 7 um diameter laser spot, a 10 ms laser pulse duration, and a 85 mW laser power were used for the microdissection. Cells from cortex and hippocampus were included as positive controls, because those areas have previously been reported to have high levels of c-Fos and lacZ expression (Smeyne et al., 1992b and 1993). Total RNA was extracted from the LCM samples with the Total RNA Microprep Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols. RT-PCR using primer pairs for c-Fos and β-actin was performed by using the OneStep RT-PCR Kit (Qiagen, Valencia, CA) as described above. The total RNA extract from each LCM sample of single cells was used in each RT-PCR reaction (15-20 single cells for β-actin and 30-40 single cells for c-Fos).
Subsequent to X-Gal staining, the sections were washed in DEPC-dH2O for 5 min and dehydrated by the following procedure: 70% ethanol for 30 s, 95% ethanol for 30 s, 100% ethanol for 30 s, xylene for 5 min, air dried in a fume hood for 15 min, and then desiccated with Drierite (W.A. Hammond Drierite, Xenia, OH) for 1 h. X-Gal stained cells in the CeA, cortex and hippocampus were microdissected using the PixCell II LCM system (Arcturus, Mountain view, CA). A 7 um diameter laser spot, a 10 ms laser pulse duration, and a 85 mW laser power were used for the microdissection. Cells from cortex and hippocampus were included as positive controls, because those areas have previously been reported to have high levels of c-Fos and lacZ expression (Smeyne et al., 1992b and 1993). Total RNA was extracted from the LCM samples with the Total RNA Microprep Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols. RT-PCR using primer pairs for c-Fos and β-actin was performed by using the OneStep RT-PCR Kit (Qiagen, Valencia, CA) as described above. The total RNA extract from each LCM sample of single cells was used in each RT-PCR reaction (15-20 single cells for β-actin and 30-40 single cells for c-Fos).
==Houpt Lab LCM Operating Procedure==
===Startup===
1. Turn on power for Samsung monitor, VGA up-converter,and MTI camera
2. Turn on power for PixCell II Microscope
3. turn on power for Controller (very stiff switch!)
===Dissection===
1. Place slide on stage, covering up the "I" shaped vacuum port
2. Engage vacuum on Controller, to secure slide to stage
3. Use "Prep strip" on slide to clean up surface of section
3. Pick up CapSure cap in swing arm, and swing over to place cap on top of slide (by grabbing the "beer can")
4. Turn laser "interlock" key clockwise from noon to 3 o'clock: this unlocks the laser
5. Press "Laser Enable" to get targeting laser. If you decrease microscope lamp illumination, you should see the red dot of the targeting laser. (It will be more less diffuse until the laser is focused).
6. Adjust size and focus of the laser spot with the controls on the left side of the laser mounted on the Microscope
7. Adjust the duration and power parameter of the laser pulse on the Controller.
* use the upper up-and-down arrows to select pulse parameter
* use the lower up-and-down arrows to set the parameter value
* Spotsize is set with the controls on the laser in previous step
* Temperature is read-only
8. Use microscope joystick to maneuver red dot over target region on the slide
9. Press the laser button to fire the laser. Can fire repeatedly to ensure collection.
10. Use microscope joystick to maneuver to an adjacent (or distant) spot to collect more samples.
11. Move cap over to "cap holder" on right of microscope.
[[Category:Protocols]]
[[Category:Protocols]]