Does the proteinase K digestion condition in the in situ hybridization kit need to be adjusted according to tissue type?

For E10.5 whole embryos, 10 μg/ml proteinase K for 15 minutes is indeed too strong and tends to cause dissociation. Whole embryos differ from sections: the tissue is intact, internal penetration is slow, but the epidermis is more fragile. It is recommended to first reduce to 2-5 μg/ml at room temperature or 37°C for 8-10 minutes with gentle shaking; the heart cavity and cartilage regions can be extended by 2-3 minutes as appropriate, but whole embryos cannot be precisely controlled locally, so err on the shorter side rather than longer. After completion, wash with PBS containing 0.1% Tween-20, then re-fix with 2% PFA for 20 minutes, which can significantly reduce embryo dissociation. Uneven signal is mostly related to insufficient or excessive digestion; a gradient of 4, 6, 8, and 10 minutes can be tested.

Rapid consumption of the hybridization buffer supplied with the kit is a common issue. You can purchase the same brand in bulk separately, or prepare it in-house according to the formulation: 50% formamide, 5×SSC, 0.1% Tween-20, 50 μg/ml yeast tRNA, 50 μg/ml heparin, 5 mM EDTA, adjust pH to 6.0–6.5, aliquot and store at -20°C. In whole-mount FISH, heparin can reduce background. Before switching to the self-prepared solution, run a parallel experiment with the same probe first to confirm that the signal does not decrease significantly. If you still rely on the kit, it is advisable to contact the distributor to check whether there is a catalog number for a replenishment solution; it is usually cheaper than a full kit, but the lead time may be 1–2 weeks.

Hybridization buffer can theoretically be recovered, but whole-mount samples are large and will carry away a substantial amount of RNase and cell debris, and the formamide and salt concentrations are also prone to evaporation and change. I recommend reusing the same batch with the same probe once, at most twice; for the second use, supplement with 10-20% fresh hybridization solution and re-denature the probe at 65-70°C. Do not introduce weak signal or background problems into the next batch of samples just to save 8 ml. A 50 ml volume used once at 8 ml can actually be used 5-6 times; if you have many samples, it is best to buy a large package or prepare it yourself, and do not treat the number of reuses as the main way to save.