Laser Capture Microdissection

Usage Fees:  $15 per hour.  Training and support, while limited, will be provided by Core Facility staff.

Note:  The LCM system is an end-user instrument.  All sample preparation, capturing and post-processing analysis must be performed by the end-user.  All reagents, including caps, must be purchased by the end-user.  The Research Core Facility does not keep caps in stock.

Scheduling:  Go to www.brownbearsw.com/cal/rcflcm.  The password will be provided after training has been completed.

Recommended Products for Laser Capture Microdissection:

The LCM system is an end-user instrument.  All sample preparation, capturing and post-processing analysis must be performed by the end-user.  All reagents, including caps, must be purchased by the end-user.  The Research Core Facility does not keep caps in stock.

Products	Catalog#	Size	Price
CapSure HS Caps	LCM0214	32 caps	$464
CapSure Macro Caps	LCM0211	48 caps	$298
GeneAmp 0.5ml Tubes	N8010611	1000 tubes	$205
Plain, Uncoated 1x3" Slides

CapSure HS LCM Caps:

• Optimal for smaller numbers of cells (capture several hundred cells).
• Contain a ridge to keep the active capture area 12 μm off the surface of the sample.
• Fit directly onto included ExtracSure devices to minimize the dilution of biological molecules extracted from captured cells; only 10 ul of extraction volume needed.

CapSure Macro LCM Caps:

• Provide the maximum area for capturing (capture several thousand cells).
• Fit directly onto 0.5 ml microcentrifuge tubes for extraction of biological molecules from captured cells.

GeneAmp Thin-Walled Reaction Tubes:

• ExtracSure devices (used with HS caps) and macro caps fit directly onto these tubes for extraction of biological molecules from captured cells.

Plain, Uncoated Glass Slides:  

• Do not use coated slides - tissue will adhere too strongly, making pickup difficult.
• Do not use membrane slides - these are optimized for UV cutting, which our system is not equipped with.

 

Important Considerations Prior to Beginning the LCM Process:

• If the target nucleic acid is RNA, RNase-free conditions should be observed at all times during handling of tissues and sections.
• Make sure that your tissue is cut to the appropriate thickness:  5 to 10 μm sections for paraffin embedded tissues or 10 to 15 μm sections for frozen sections.
• Make sure that your tissue section does not contain folds or uneven spots that will prevent the cap from sitting flush on the tissue. 
• Use a histological stain that facilitates cell identification but also preserves the integrity of the biomolecules of interest.  The following is not a complete list but are recommended for some applications:

Hematoxylin (Harris not recommended) Eosin Nuclear Fast Red Methyl Green Cresyl Violet (recommended when performing PCR) Toluidine Blue

• Dehydration is mandatory for LCM.  Samples with any residual moisture will be subject to hydrostatic forces, making it difficult to separate the target tissue from the slide and from the surrounding tissue.  Poorly dehydrated samples will appear more translucent and will usually have a shinier surface than thoroughly dehydrated samples.
• For optimal capture, use caps that are not damaged or old.  

Example Staining and Dehydration Protocol (from the Paradise PLUS FFPE LCM Staining Kit):

• Label 10 plastic slide jars as follows: 

a.    Xylene_1 b.    Xylene_2 c.    100% ethanol_1 d.    95% ethanol_1 e.    75% ethanol_1 f.    Nuclease free water g.    75% ethanol_2 h.    95% ethanol_2 i.    100% ethanol_2 j.    Xylene_3

• Using the LCM-certified solutions provided (if using the Paradise PLUS Staining Kit), fill the labeled plastic slide jars with 25 ml of the appropriate solution. 
• Remove up to four slides from the slide box or from the –70°C freezer, and place in a 50–60°C oven for 2 minutes. 
• Place the slides in plastic slide jar “a” containing xylene for 2 minutes. Invert jar gently. 
• Transfer the slides to plastic slide jar “b” containing xylene for 2 minutes. Invert jar gently. 
• Transfer the slides to plastic slide jar “c” containing 100% ethanol for 2 minutes. Invert jar gently. 
• Transfer the slides to plastic slide jar “d” containing 95% ethanol for 1 minute. 
• Transfer the slides to plastic slide jar “e” containing 75% ethanol for 1 minute. 
• Transfer the slides to plastic slide jar “f” containing nuclease free water for 30 seconds. 
• Using an RNase free pipette tip, apply 100 μL of the Paradise PLUS Staining Solution so that it covers the entire section. Stain for 15-45 seconds at room temperature. Tap off excess stain before proceeding with the following steps. 
• Transfer the slides to plastic slide jar “g” containing 75% ethanol for 30 seconds. 
• Transfer the slides to plastic slide jar “h” containing 95% ethanol for 30 seconds. 
• Transfer the slides to plastic slide jar “i” containing 100% ethanol for 1 minute. 
• Transfer the slides to plastic slide jar “j” containing xylene. Hold slides in xylene until ready for microdissection. The minimum incubation in xylene should be 5 minutes, or up to a maximum of ~2 hours. Do not use an oven to dry.
• Place the slides on a Kimwipes cleansing wipe to dry in the hood for five to ten minutes prior to LCM. LCM should be performed within 2 hours after removal from xylene. 
• Discard all used staining and dehydration solutions according to standard procedures. 

Notes:

• Carry out the Staining and Dehydration segment of the protocol in a fume hood.
•  Wear clean disposable gloves.
•  Xylene jar “a” must be changed after processing up to a maximum of 4 slides.
•  75% Ethanol jar “e” must be changed after processing up to a maximum of 4 slides.
• When utilizing multiple slides for capture, place any slides not on the LCM instrument stage in a box containing desiccant to reduce the accumulation of moisture.