Table 4. Troubleshooting guide.

Step	Problem	Possible Reasons	Solution
13A	No staining	Inadequate fixation; suboptimal antibody concentration.	Use fresh 4 % formaldehyde solution; fix samples for 2 h on ice with gentle agitation; use fixative at 10× volume of tissue to be fixed; alternatively, test different fixative reagents; optimise antibody concentration; test alternative antibodies.
	fluorescent speckles	Secondary antibody forms precipitates.	Spin secondary antibody in a refrigerated benchtop centrifuge at top speed before use.
	Weak staining	Insufficient tissue penetration of antibodies; antibody concentration suboptimal; photobleaching.	Increase incubation time; optimise antibody concentration; keep samples in the dark (e.g. wrap containers in tin foil); mount samples in antifade solution.
13Bviii	No staining	Inadequate fixation; suboptimal antibody concentration.	Use fresh 4 % formaldehyde solution, fix hindbrains for 2 hours on ice with gentle agitation; use fixative at 10× volume of tissue to be fixed; optimise antibody concentration; test fresh batches of primary or secondary antibodies.
	High background	Residual endogenous peroxidase activity; insufficient washing after antibody incubation; overdeveloped reaction.	Check if correct bleaching with hydrogen peroxide was performed before staining; increase frequency of antibody washes; observe HRP reaction under a stereomicroscope every 5 minutes.
	Weak staining	Insufficient tissue penetration of antibodies (E12.5 onwards); not enough time for HRP reaction; suboptimal antibody concentration.	From E12.5 onwards, use vibratome sectioning followed by immunolabelling; allow at least 30 min for HRP reaction, observe reaction under a stereomicroscope every 5 minutes; incubate with new developing solution; optimise antibody concentration.
13C	Hindbrain separates from agarose	Excess liquid attached to hindbrain at embedding; incorrect settings for sectioning (e.g. alter sectioning speed).	Carefully blot liquid from samples with clean tissue paper before embedding; optimise speed and vibration amplitude of vibratome.
13D(i,ii)	Tissue degeneration	PBT was used instead of PBS before tissue fixation.	Use 10% FBS in PBS to block non-specific interactions before adding conditioned medium. Use PBS to wash unbound AP-fusion proteins before tissue fixation.
13Dviii	Weak staining	Not enough time for AP reaction; suboptimal AP-fusion protein concentration.	Observe under a stereomicroscope at 5 minute intervals; allow at least 30 min for AP reaction; incubate with fresh developing solution; spot conditioned medium on nitrocellulose filters and develop to verify AP-fusion protein expression.
14C	Air bubbles in mounted sections.	Trapping of air bubbles when adding the coverslip.	Carefully lower the coverslip onto the section to avoid trapping air bubbles.