Immunology and 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.

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.

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.