Laser Capture Microscopy (LCM)

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Supplies for LCM

Cryo-Jane System for Adhesive Slides

Instrumedics Inc. 5918 Evergreen Blvd. St. Louis, Mo 63134 Phone: 314-522-8671 Fax: 314-522-6360 email: sales@instrumedics.com

(last ordered in 2007, so prices probably different)

Adhesive-Coated Slides (125/box) #475208 $175

Tape Windows (400/roll) 475214 $45

Protocol for Amygdala Regions

Reference Kwon et al, 2008, PMID 18374904

The frozen brains were sectioned between -2.30 and -3.14 mm from bregma (Paxinos and Watson, 1986) at 5 µm thickness and mounted on slides in a –20 ºC cryostat using the CryoJane system (Instrumedics, Hackensack, NJ). The frozen brain sections were defrosted at room temperature for 30 sec, then stained and dehydrated by the following procedure: 75% ethanol for 30 sec, DEPC-treated deionized H2O (DEPC-dH2O) for 30 sec, Histogene Staining solution (Arcturus, Mountain View, CA) for 3 min, DEPC-dH2O for 30 sec, 75% ethanol for 30 sec, 95% ethanol for 30 sec, 100% ethanol for 30 sec, 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. LCM parameters were 15 or 30 µm diameter laser spot, a 10 ms laser pulse duration, and a 65 mW laser power. Multiple laser shots (150-200) were required to transfer each amygdala region. Following dissection, tissue sections were stained with methyl green (Vector Laboratories, Burlingame, CA) and coverslipped to verify the specificity of the dissection.

Total RNA was extracted from individual LCM samples with the Total RNA Microprep Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols. RT-PCR was performed by using the OneStep RT-PCR Kit (Qiagen, Valencia, CA).

Protocol for X-Gal Stained Cells

Reference Kwon and Houpt 2010 PMID 19925827

In order to confirm mRNA expression and preservation in β-Gal-positive cells, we performed RT-PCR after LCM of single cells. Transgenic mice were injected with LiCl (0.15 M, 40 ml/kg, i.p.). One hour later, they were anesthetized with sodium pentobarbitol, decapitated, and the brains were dissected. The brains were emmersed in M-1 Embedding Matrix (Shandon, Pittsburgh, PA), immediately frozen with dry ice, and stored at –80 ºC freezer until used. The frozen brains were sectioned at 8 µm and mounted on slides in a –20ºC cryostat using the CryoJane tape transfer system (Instrumedics, Hackensack, NJ). The sections were defrosted at room temperature for 30 s and then briefly washed with DEPC-dH2O. The brain sections were fixed in 70% ethanol at room temperature for 5 min and then washed with DEPC-dH2O for 5 min. Brain sections were then stained as above in X-Gal reaction buffer containing 1 mg/ml of X-Gal in DEPC-dH2O. The reaction was performed for 16 h at 37ºC in darkness.

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).