Put the slides 30 min in the incubator at 60ºC

Wash 3x3 min in xylene

Wash 1x10 min in ethanol 100%

Wash 1x10 min in ethanol 95%

Wash 1x5 min in ethanol 70%

Wash 2x5 min in PBS 1X

Incubate slides in citrate buffer 10mM, pH 6 in a boiling water bath (95ºC) for 20 min

Let sections cool down for 20 min at room temperature (RT)

Wash 3x5 min in PBS 1X-Triton (0.5%)

Put wet paper in a black box for immunohistochemistry

Gently dry and circle sections with the hydrophobic pen ImmEdge Vector H 4000 without touching the tissue

Add 200 uL/section of blocking solution (5% NGS (in PBS 1X) (200uL/section) + 0.1% Triton (in PBS 1X)) 1h at RT

Gently wipe off the water ofthe slides

Put 200ul/section of 1ary antibody in 2% NGS + 0.1% Triton overnight at 4ºC (cold room)

Wash 3x5min in PBS 1X-Triton (0.5%)

Gently wipe off the water ofthe slides

Put 200ul/section of 2ary antibody in 2% NGS + 0.1% Triton + Hoechst/DAPI for 1 h at RT

Wash 3x5min in PBS 1X

Mount sections with fluorescent mounting medium. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles

Let dry the slides on the tray in the hood for 5 minutes and store at 4ºC asap