LCM - Sample Preparation

What you will need for microdissection

• Special slides with membrane for mounting and staining sections (part # 50103). For RNA work you will need the individually wrapped RNAse free slides (part # 50102)
• Collection tubes with specialized caps (part # 50202
• Regular glass slides for supporting (Also made RNAse free for RNA work)
• A blank CD for copying your images
• Extraction buffers, gloves, dry ice, Container with desicant if your samples were stored at -80 or on dry ice.

The special slides and tubes can be purchased from:

Molecular Machines & Industries, Inc.
PO Box 560728
Rockledge, Florida 32956-0728
Phone: + 603 629 9536
Fax: +1 321 978 0304
Sales_us@molecular-machines.com
www.molecular-machines.com

All orders for consumables and systems should be faxed to the number shown above. Please inform also your administration and purchasing department that checks need to be sent to this address.

For price information see consumables.

Precautions to take when preparing samples for laser microdissection

• Recovery of material is best using frozen sections. If your samples are fixed in formalin you will need special methods for recovering RNA/DNA.
• When trying to extract RNA all precautions must be taken to avoid RNAse activity at every step to ensure success. These include;
  1. use of RNAse inhibitors at each step
  2. storing samples at -80
  3. making sure all surfaces, brushes, etc. that come in contact with sections are RNAse free by wiping with 100 % ethanol or an RNAse inhibitor solution
  4. Try to finish staining on the same day as cutting in order to avoid freezing and thawing
  It would be helpful to check on the quality of your preparation by using some of the stained slides to analyze the integrity of RNA in your preparations. This will save a lot of time and effort downstream spent in cutting and analysis.

Sectioning
Immediately after collection freeze samples in OCT and store at -80 until you are ready to cut and stain. Try to finish staining on the same day as cutting. Also make sure that you don't freeze thaw. Keep the tissue block in cooled cryostat. Before cryosectioning, make sure all surfaces, brushes, etc. that come in contact with sections are RNAse free by wiping with 100 % ethanol. Cut 8-10 micron sections (the sections can be as thick as 20 microns if needed). The sections are then mounted on the special microdissection slides that have a thin plastic film attached on one side of a metal frame so that one side is level with the frame while the other side forms a trough/depression (see illustration). Pick up cut section by touching it to the side of the slide that has the membrane attached, so that the section is level with the slide surface, and not in the depression (Note, it is important that the section be mounted on the correct side of the membrane to avoid problems during microdissection). Briefly warm the section by holding the slide at room temperature until the OCT melts (few seconds). The slides should be kept at room temperature prior to sectioning to make sure that there is no condensation on the plastic film since this may prevent the bonding of tissue to the film.

Staining
For gene expression work use DEPC-treated or RNAse free water for making solutions. To be extra cautious add RNAse inhibitor to the staining solutions (recommended). Instead of dipping slides in stains, pipette the solutions on the tissue specimen (to avoid wasting RNAse inhibitors). Do not reuse staining solutions. DNA and protein are more stable, and such precautions are not needed.

95% ethanol -15-45 sec (This also serves as a fixation step)
70% ethanol -15 sec
Water -15 sec
Hematoxilin -20-30 sec (If over stained, dilute for better staining)
Water rinse 2x -15 sec
Bluing soln -few sec
70% ethanol -15 sec
Eosin Y -5 sec
95% ethanol -15 sec
100% ethanol -15 sec
100% ethanol -15 sec
Drain off the ethanol
Xylene -60 sec

Air dry for few min, Place a dry glass slide so that the section is sandwiched between the plastic film and glass slide. To protect the RNA, the glass slide should be made RNAse free before it contacts the section, either by baking in the oven or by wiping first with an RNAse inhibitor solution, followed by 100% ethanol. If not carrying out microdissection on the same day, store the slides in 50 ml falcon tubes and keep at -80 degrees centigrade. A few hours before dissection, place the slides in a bottle with desiccant so that they remain dry during the time it takes for them to warm up to room temperature. It is essential to minimize hydration to avoid loss of RNA. If dissecting on same day as sectioning and staining, you can leave the slides at room temp.

Slides can also be immunostained using the HRP or Alkaline Phosphatase conjugated antibodies prior to staining to facilitate identification of specialized cells. Alternately, fluorescent markers may be used since the microscope is equipped with blue (DAPI), green (FITC/GFP) and red (Rhodamine/RFP) filters.

The protocol described above describes preparation for RNA extraction, it can also be used for DNA and protein extraction. Details specific for individual needs can be discussed during initial consultations.