Before you start

Plan section thickness (often 10–20 µm for spinal cord/brain) and section placement across slides.

Reagents and materials

Totals are for 10 slides (+10% extra). Per-slide amounts are in the middle column.

Reagent	Per slide	Total
4% PFA (perfusion and post-fix)per tissue for post-fix	2 mL	2 mL
30% sucrose in PBSper tissue	5 mL	5 mL
OCT compound	1 mL	1 mL
Blocking/permeabilisation buffer (5% normal serum, 0.3% Triton X-100 in PBS)	0.3 mL	0.3 mL
Primary antibody in blocking buffer	150 µL	150 µL
Secondary antibody in blocking buffer	150 µL	150 µL
DAPI (1 µg/mL)	0.3 mL	0.3 mL
Antifade mounting medium	30 µL	30 µL

Adjust to pH 7.4 and autoclave. This PBS is Ca²⁺/Mg²⁺-free (use it for detaching cells).

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

Fix
Perfuse with PBS then 4% PFA (as approved), dissect and post-fix in 4% PFA for 4–24 h at 4 °C.
Cryoprotect
Transfer to 30% sucrose at 4 °C until the tissue sinks (usually 24–48 h).
Embed and freeze
Embed in OCT in a mould and freeze on dry ice or in isopentane cooled on dry ice. Store at −80 °C.
Section
Equilibrate the block to the cryostat temperature (about −20 °C), cut 10–20 µm sections onto charged slides and air-dry 30–60 min.
Rehydrate
Wash 3 × 5 min in PBS to remove OCT.
Block and permeabilise
Draw a hydrophobic barrier and block for 1 h at room temperature.
Primary antibody
Incubate overnight at 4 °C in a humid chamber.
Secondary antibody
Wash 3 × 5 min, then incubate with secondary antibody for 1–2 h at room temperature in the dark.
Counterstain and mount
Wash, DAPI 5–10 min, wash, mount with antifade and coverslip.

Antigen retrieval (e.g. citrate pH 6.0) may be needed for some antigens after PFA fixation.
Myelin and lipofuscin autofluorescence in CNS tissue can be reduced with commercial quenchers.

Problem	What to check or change
Sections fall off	Use charged slides, dry longer, avoid harsh washes.
Ice crystal artefacts	Complete cryoprotection and freeze rapidly.
High background	Longer blocking, titrate antibodies, include secondary-only controls.

PFA in a fume hood; cryostat blades are very sharp — use the blade guard.