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Sequencing and Imaging

1. In-Situ Sequencing

Complete Cycles 1-10 using the automated fluidic system.

2. Morphology Staining

Goal: Visualize nuclei and cell boundaries.

Reagent Mix: * DAPI: 5 μg/mL * WGA-Alexa 488: 5 μg/mL

Procedure: 1. Wash: PBS-T (0.05%) for 2 min. 2. Stain: Pump DAPI/WGA mix. Incubate 15 min. 3. Wash: Pump PBS. 4. Image: Scan in DAPI and FITC channels. 5. Clean Lines: Pump 4X SSC then PBS-T (0.1%) to remove sticky WGA.

3. H&E Staining (Post-Sequencing)

Compatible with Flow Cell (No Xylene).

  1. Hematoxylin: 2-5 min.
  2. Rinse: DI Water.
  3. Differentiation: 0.5% HCl in 70% EtOH (2-5 seconds). Critical timing!
  4. Bluing: Scott's Tap Water Substitute (1 min).
  5. Eosin: 30s - 1 min.
  6. Clear: 95% Ethanol (Do NOT use Xylene).
  7. Image: Brightfield.