Stimulated emission depletion microscopy (STED)

See attached protocol file (better numbering)

Immunohistochemistry for STED on free-floating vibratome sections

Prior to immunohistochemical staining, the animals were perfused with 1xPBS and heparin (20 U/ml), followed by 4% PFA, and the brains were removed from the skull and post-fixed overnight in 4% PFA. 

Tissues were stored in 1xPBS at 4ºC and sectioned into 50 µm thick sections using the HM650V Vibratome (Thermo Fisher Scientific). For long-term storage, use sodium-azide (NAN₃).

Sections were stored in 1xPBS in Parafilm-sealed 24-wells-plates at 4ºC. It is advised to proceed with the staining as soon as possible.

• Use shallow 12-well glass plates for incubation with blocking buffer and antibodies (wells lined with PAP-pen) (you can also use plastic 12-well-plates, but you’ll need a larger volume of your antibodies).

• Use regular plastic 12-well-plates + grid-inserts for washing.

• Use a clean brush for transferring sections from the different well-plates.

Part 1

Perform all incubation and washing steps horizontally in a humified dark chamber, shaking 

1. Wash sections 3 x 5 min with PBS at room temperature (RT)

2. Permeabilization in a 12-well-plate with grid inserts, containing 1xPBS + 0.2% Triton X-100) for 1h at RT 

3. Blocking in 12-well glass plate with 10% serum (species in which the secondaries were raised, we used goat secondaries, therefore normal goat serum (NGS)) + 1% BSA in PBS + 0.2% T_X for 1h at RT 

4. Incubation with primary antibodies in 5% NGS + 0.05% Triton X-100 + 0.01% NAN₃, in a new 12-well glass plate (transfer sections using a brush, clean between each section), for 72h at 4ºC

Part 2

Perform all incubation and washing steps horizontally in a humified dark chamber, shaking

1. Transfer sections to a 12-well-plate + inserts containing 1xPBS, wash 3x for 10’ at RT

2. Incubation with secondary antibodies in 5% NGS + 0.05% Triton X-100 + 0.01% NAN₃ in a 12 well glass plate for 48h at 4ºC

3. Transfer sections to a 12-well-plate + inserts containing 1xPBS, wash 5x for 10’ at RT 

Note: if you know that the primary antibodies cross-react, separate the incubation steps: repeat primary and secondary incubation steps (including washing), for the second primary/secondary combination. 

1. Using a basin with 1xPBS transfer the sections to glass slides, let them dry

2. Coverslip with ProLong Gold Antifade and Marienfeld Superior 1.5H high precision coverslips 

3. Store in the dark at 4°C

4. Proceed with the imaging as soon as possible