What are the optimization points for a new tissue type?

1. Digestion (Right before probe hybridization)
Optimization Focus: Optimize digestion time only.

Harder/Denser Tissues: If tissues are expected to be harder to penetrate, 15 to 20 minutes of digestion at room temperature is typically sufficient.

Fragile Tissues: If the tissue appears fragile, keep the digestion time to 10 minutes or less.

Fixation Tip (Small Immune Cell Retention):
Adding 0.007% glutaraldehyde to 4% PFA and fixing the tissue at 30°C significantly improves the retention of small immune cells, which is highly recommended for this project.

Phenotypes of Over-Digestion:

Tissue structural breakdown / tearing

"Donut-shaped" DAPI staining

Fuzzy or blurry DAPI staining

2. Clearing (Final step)
Impact on Performance: Inadequate clearing directly hinders sequencing probe penetration, leading to lower signal quality.

Remediation: Tissues can undergo re-clearing if optimal clarity is not achieved after the initial run.