Histology

Fixation and processing of embryos

• • Relaxation

  • General-purpose 4% formaldehyde fixation

  • Biphasic fixation

  • Fixation for immunostaining against catecholamine neurotransmitters

  • Removal of vitelline membrane after fixation

Immunostaining for Helobdella embryo [CSH Protocols] [addendum]

Arnold's silver staining for visualizing cell-cell boundaries on the surface of the embryo [CSH Protocols]

Phalloidin labeling for visualizing F-actin

DAPI or Hoechst staining for visualizing DNA (chromosome)

Whole-mount in situ hybridization for Helobdella embryo [CSH Protocols]

• • Probe synthesis [standard protocol + hydrolyzed probe protocol]

  • Standard protocol [simplified from the CSH Protocols version, equally good results nevertheless]

  • Double in situ protocol

  • Fluorescent (double, triple or quadruple) in situ protocol

  • Hybridization Chain Reaction (HCR)

Enzymatic color reactions [guide for selecting an appropriate substrate]

• • HRP: DAB (brown) [due to its high carcinogenic toxicity, DAB is banned from our lab]

  • HRP: DAB + nickel (black)  [due to its high carcinogenic toxicity, DAB is banned from our lab]

  • HRP: Nova Red (red) [to use, see manufacturer's instruction: NovaRED (Vector Labs, SK-4800)]

  • HRP: tyramide signal amplification (various fluorophores and haptens)

  • AP: NBT/BCIP (blue/purple)

  • AP: BM Purple (Roche 11442074001) (blue/purple)

  • AP: BCIP (turquoise)

  • AP: INT/BCIP (orange)

  • AP: Fast Red or Vector Red (bright red) [to use, see manufacturer's instruction: Fast Red (Roche 11496549001 or Sigma F4648), Vector Red (Vector Labs SK-5100)]

Enzyme inactivation for double enzymatic labeling

Lauter et al. (2011 Neural Dev. 6, 10) found acid glycine treatment is effective for both alkaline phosphatase and peroxidase. To do this simply incubate specimens in 0.1 M glycine pH 2, 0.1% Tween-20 for 10 minutes at rooom temperature. Acid glycine is likely working by stripping antibodies from the sample rather than killing enzyme.