Take out sections from -80 and place them in an incubator/plate for 0h 20m 0s at 37°C. This step is performed to ensure tissue attachment to the crystal slides.

2. Draw a hydrophobic barrier on each slide using ImmEdge®Hydrophobic Barrier PAP Pen ^®Hydrophobic Barrier PAP Pen and let dry for about 0h 10m 0s minutes. This will prevent buffers from escaping the tissue area.

To initially rehydrate and permeabilize the tissue, place the slides in a slide jar containing the permeabilization solution with shaking for 0h 30m 0s

.

To prevent unspecific binding, incubate brain sections in permeabilization solution containing 5% (v/v) and 1Mass / % volume for 1h 0m 0s at Room temperature.

When blocking is finished, decant the buffer (no washing is required) and incubate primary antibodies in antibody buffer for 3h 0m 0s at Room temperature according to table 1 .

A	B	C	D	E
Antibody	Company	Reference	Specie	Dilution
Gfap	Invitrogen	13-0300	Rat	1:500
Iba1	Wako	019-19741	Rabbit	1:500
NeuN	Millipore	ABN91	Chicken	1:300

Table 1. Primary antibodies

When primary antibody incubation is finished, wash the sections 0h 5m 0s with 0.05% (v/v) in PBS.

Incubate secondary antibodies in 0.1% (v/v) for 1h 0m 0s at Room temperature according to table 2.

A	B	C	D	E
Antibody	Company	Reference	Channel	Dilution
Goat anti-rat	Invitrogen	A11006	488	1:500
Goat anti-rabbit	Jackson	111-165-003	Cy3	1:500
Goat anti-chicken	Invitrogen	A32933	647	1:500
Dapi	Invitrogen	D3571	405 (Dapi)	1:5000

Table 2. Secondary antibodies

When secondary antibody incubation is finished, wash the sections 0h 5m 0s with 0.05% (v/v) in PBS. Follow this with 0h 5m 0s washes with PBS to remove all detergent traces.

Clean the remaining buffer on the slides using absorbent tissue and mount the sections with a drop flo.