Workflows & Protocols: Connecting Microscopy and Molecular Biology in Neuroscience

Report on a Leica LMD user workshop held in Brazil

December 04, 2015

During this second Leica laser microdissection (LMD) Workshop in Brazil, hosted by the Federal University of Paraná (UFPR) and held by Dr. Falk Schlaudraff, Product Manager at Leica Microsystems, the Leica LMD system was introduced to new users. In addition, participants of the first LMD workshop at the Centro de Energia Nuclear na Agricultura/USP (CENA) could deepen their knowledge about laser microdissection.

Fig. 1: Different fresh and cryo-stored samples in a cryostat equilibrating for the optimal cutting temperature

The main topic during this course was how to apply laser microdissection in neuroscience. Leica specialists demonstrated why laser microdissection is a suitable techniques for brain investigation as it allows to separate distinct brain layers or even to isolate individual neurons.

After a brief introduction into the world of laser microdissection, its various options, different applications, tips & tricks, the attendees got the chance to operate the system themselves. In addition, Leica Microsystems offered trainings on different sample preparation techniques.

Every participating scientist got the chance to test and verify the entire workflow from the native sample to the desired pure target cell(s) of interest in an inexpensive standard PCR tube.

Fig. 2: Specimen Overview of a murine midbrain section, unstained, 60 µm, before (left) and after (right) isolation of the Substantia Nigra with a Leica LMD7000 system.

Beside the training of different sample preparation techniques some scientists brought their own samples for dissection. One task was to collect the whole substantia nigra from an unstained mouse brain (Fig. 2).

"After the evaluation of some other systems the participants were surprised by the power of the Leica LMD system," Falk said. "Our laser allows to dissect the 70 µm unstained section with a single cut, by the speed and option to collect big areas covering multiple fields of view. In this way, it was possible to collect the desired brain area from all provided samples in very little time. Such large laser microdissected areas of unstained and well-defined brain layers are perfect starting material for proteomics and metabolomics."

The second task was to dissect individual cells - instead of collecting a whole brain area such as substantia nigra or smaller areas like hippocampal layers. Both tasks, big areas and single cells, can be easily accomplished with the Leica LMD system by either marking the target or directly dissecting it with the laser. Both methods have their advantages as agreed by the group.

Marking the material allows corrections, display information like the marked target area size in µm² and delegating the targets into one or different collectors. "Dissecting live, without the need of drawing a line first – which is unique for the Leica LMD systems – is very fast and also entertaining," Falk stated.

The real time application Move+Cut can be combined with the drawing by re-dissecting challenging tissue parts or recovering not completely dissected areas.

Fig. 3: Single cell collection example. Left: a single neuron is marked for dissection. Center: same area after dissecting the neuron (both: 63x objective, mouse brain section 10 µm, stained with cresyl violet). Right: seven collected cells can be visualized quickly in the collection cap with a 4x objective.

The quick and easy-to-learn approach of dissecting target cells, individual cells or larger areas independent of their size, shape, amount and distribution on the slide, underlined the flexibility of the Leica LMD system for various applications. After the workshop, all participants were able to operate the system for their individual task within minutes and felt comfortable collecting their targets.

Beside the manual process of laser microdissection, the Leica AVC module (pattern recognition) was introduced. This module helps to speed up the process to collect individual cells and requires a minimum of interaction with the system.

During the sample preparation workshop several organs of a mouse were prepared for dissection. These samples require special protocols in order to preserve the molecular content without negative effects as they will be investigated after laser microdissection.

Fig. 4: A new user starts to get confident with the system hunting for single cells. Beside the manual process of laser microdissection the Leica AVC module (pattern recognition) was introduced as well, speeding up the time to collect individual cells dramatically and also requires a minimum of interaction with the system.

Protocol steps in brief for fixation and cresyl violet staining of murine brain sections [1]:

  1. Ensure clean (e.g. RNAse-free) and sterile working conditions.
  2. Cool the cryostat to -35°C.
  3. Prepare the whole brain freshly and in case needed trim it.
  4. Place the brain on the holding device of the cryostat with OCT.
  5. Equilibrate the brain for 1 hour in the cryostat at -35°C.
  6. Equilibrate the brain for 45 minutes at -19°C.
  7. Adjust the cryostat to apply suitable cuts.
  8. Cut the brain (50 µm sections) to achieve a plain surface near the region of interest.
  9. Cut some sections at 10 µm (if the sections roll, decrease the temperature and equilibrate again for 15 minutes, if the section get ruptures, increase the temperature and equilibrate again for 15 minutes)
  10. Prepare one or more flat sections for mounting (if needed use a brush to stretch the section(s)).
  11. Use a membrane slide from room temperature and place the membrane side carefully on top of the frozen section (the section will melt to the membrane).
  12. Place the slide with the mounted section(s) into a 50 ml Falcon tube with 75% EtOH at -20°C for 2 min (75% EtOH should be prepared and stored at -20°C over night).
  13. dip the slide into a 50 ml Falcon with 1% cresyl violet in 75% EtOH for 1 min
  14. Dip (ca. 4 sec) the slide into a ascending EtOH row: 75% EtOH, 90% EtOH and finnaly 100% EtOH, leave it at 100% for 30-60 sec.
  15. Dry the slide in a 50 ml Falcon tube with a silica bag in the conus for at least 20 min.

While this procedure is straightforward, other types of tissues such as plants can be more tricky or even easier to dissect. This will be topic for the next article.

Fig. 5: Participants of the second LMD Workshop

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