Laser Capture Microdissection

The Arcturus XT Laser Capture Microdissection (LCM) Instrument provides an automated approach to laser microdissection of individual cells or multi-cellular structures from slides containing tissue sections. It consists of a Nikon Eclipse Ti-E inverted microscope with a solid-state near IR laser. 2x, 10x, 20x, and 40x objectives are available for use. The stage is formatted to accept up to three 1"x 3" slides.

Principles of LCM:  Laser capture microdissection (LCM) is a method used to separate individual cell types from within complex tissues.  Specifically developed caps coated with a thermoplastic film are placed on the region of interest.  The Arcturus XT instrument is then used to direct a low-powered infrared laser through the cap to melt the film onto the cells of interest.  These cells adhere to the cap when it is lifted from the tissue section – thus, capturing them for further analysis.  The laser does not affect the tissue sample; therefore, the quality of the nucleic acids and proteins within the sample are not compromised.

Fees

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

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

Products

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.

 

Tips

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.  

CONTACT US

Camille Abshire, MS, MDxT-(AAB), CLS (LSBME)
Research Specialist, Genomics
cabsh2@lsuhsc.edu
(318) 675-4174

 

Rona Scott, PhD
Director, CMTV Genomics Core
Associate Professor, Department of Microbiology and Immunology
rscott1@lsuhsc.edu
(318) 675-6263