Abbreviations. PB = 0.1 M phosphate buffer (pH 7.4); PAD = PB + 0.1% Tween-20 (permeabilisation/antibody diluent); NGS = normal goat serum (blocking agent for goat-derived secondary antibodies).

Tape-protected vibratome stage (3-DRAKON Lab). Applying a strip of broad adhesive tape to the metal stage before gluing the block is a lab-developed technique. The cyanoacrylate bonds to the tape surface rather than directly to the stage. After sectioning, the tape peels off cleanly in one piece, removing all glue residue with it and leaving the stage undamaged. This eliminates the need for solvent cleaning between samples and extends stage lifetime.

Blotting technique. Thorough blotting before each antibody application step (blocking, primary Ab, secondary Ab) is one of the most important variables for signal quality. The correct end-point is sections that are barely moist — no visible liquid film — but not dried out. Tilt the cup, wait for sections to settle fully, then wick remaining buffer from the cup wall and base with the torn edge of filter paper. Insufficient blotting dilutes the antibody working concentration; over-blotting causes sections to dry and crack.

Gelatin vs. agarose embedding. Gelatin (10%) is the primary choice: it provides excellent tissue support and melts away during staining without special treatment. LMP agarose (6%) is a valid alternative when the vibratome bath temperature rises and gelatin softens — agarose tolerates warmer baths. Both matrices are sectioned under the same vibratome settings.

Vibratome settings optimisation. The listed parameters (speed 1.2 mm/s, amplitude 1.4 mm, 40 µm) are starting values. Increase amplitude or decrease speed if sections tear. Reduce thickness to 25–30 µm for confocal Z-stacks; increase to 60 µm for thicker antibody-penetration assays.

Wash efficiency. Free-floating sections settle within seconds in PB — always wait for complete settling before removing the buffer, otherwise sections are aspirated and lost. Using wide, flat-bottomed cups (the 3D-printed ASA design) reduces section stacking and improves wash efficiency.

DAPI concentration and timing. 10 µg/mL for 15 min at RT gives reliable nuclear staining in 40 µm sections. Over-incubation leads to diffuse cytoplasmic background. DAPI stock (1 mg/mL in H₂O or DMSO) is stable at −20 °C in the dark for ≥1 year.

Coverglass mounting. The gravity-only lowering approach is critical for 40 µm free-floating sections — any mechanical pressure collapses the tissue. If bubbles form, allow them to migrate to the edge over 30 min at 37 °C before imaging; do not attempt to press them out.