Tissue and Slides

Tissue and Slides#

Tissue#

Tissues are quite heterogeneous, and you must know your tissue to best adapt the workflow to it. Here are some factors to consider when preparing for DVP.

Adhesion to membrane#

  • Better adhesion will allows you to perform more cycles of multiplex immunofluorescence.

  • Fatty tissues, like breast, will have less adhesion.

Autofluorescence#

  • Endogenous Fluorophores (collagens, elastins, lipofuscins)

  • Tissue type and composition, more connective tissue usually has more autofluorescence

  • Certain tissues (e.g., liver, lung, brain) are inherently more autofluorescent.

  • Older and fibrotic samples are worse than fresh or young tissue.

  • FFPE usually has higher autofluorescence than frozen sections.

  • Autofluorescence tends to overlap with FITC/Alexa488, less so with far-red dyes (Alexa647, Cy5).

Cell size#

  • Large cell sizes provide more input material in single-cell DVP and pooled DVP. For example, hepatocytes are large and full of input material.

  • Smaller cells would require pooling to readout the same number of proteins

  • See The dawn of single-cell proteomics..

Cell Density#

  • Denser tissues will have more z-axis noise (cells on top and below cell in focus).

  • Segmentation is more challenging with denser tissues

  • Single cell collection is prone to capturing overlapping cell material.

Tissue thickness#

  • Our default is 5 micrometers thick

  • Thicker cuts provide more input material for LCMS, but have noisier images

  • Thinner cuts provide less input material, but have greater signal-to-noise images.

Slides#

Laser microdissection of tissue cannot be done on glass slides, they stick too well.
Therefore we collect tissue from slides that have a membrane that can be cut.
The properties of the membrane vary, and you should consider which slide to use.

Leica offers two main formats:

  • Frame Slide, a metal frame with a membrane that “floats” in the tissue area.

  • Glass Slides, a glass slide with a membrane that is separated by a thin air pocket.

There are two main materials that make up the membrane:

  • PEN (polyethylene naphthalate)

  • PPS (polyphenylene sulfide); less autofluorescent for IF applications.

For a detailed read go to Leica: consumables-for-laser-microdissection

Preparing slides before imaging (work in progress)#

Increasing attachment with Poly-L-Lysine#

(work in progress)

Etching calibration points on the slide#

(work in progress)

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