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general_tips

Immunology and sectioning

Sectioning

  • Read the Leica sectioning handbook, it's full of tips and trouble-shooting.
  • At the microtome, orientate blocks so that the knife cuts the tissue at the softest part first, and at the broad face of the tissue section.
  • Cool the blocks before cutting, on ice. For hard samples letting the blocks sit on wet ice can soften the tissue and allow for better sections.
  • After sectioning, dry the slides at 37C overnight with slides in a vertical position, and then bake at 60C for one hour.

Immuno-staining

  • Bubbles in the mounting media: Apply the coverslip at an angle, seal the edges of the coverslip with nail polish, store the slides vertically, store the slides at -20C.
  • Non-specific speckles of signal: Ab aggregate, spin at max RPM, 4°C, 10'.
  • The first time using a new antibody, prepare slides with a serial dilution, and only try to do multiple-labelling with other antibodies after single labelled experiments have proven successful.
  • Be aware that some fluorescent agents (such as DAPI) have very broad emission spectrum and may cause problems, and others (such as FITC) bleach rapidly and may make imaging more difficult.
  • For each experiment make a slide without primaries, but with the secondaries, to check for non-specific staining.
  • For experiments with overlapping spectra or with auto-fluorescence in the tissue, make a slide with no primary, no secondary, and a slide with each primary and secondary pairing alone. These can be used with microscopes equipped with spectral detectors to get accurate results.
  • Background autofluoresence: Wavelength specific irradiation1): CuSO4 in ammonium acetate buffer or Sudan Black B in 70% ethanol2): NaBH43): Pontamine Sky Blue4). Or image in far-red instead of green/red.

Image acquisition

  • Avoid imaging at the edge of the image field (to avoid vignetting).
  • Keep the saturation controls on, and the auto-LUT off, but aim to have the image looking the same if the saturation controls are off and the auto-LUT is on.
general_tips.txt · Last modified: 2014/06/21 18:44 (external edit)