Put the slides 30 min in the incubator at 60ºC.

Wash 3x3 min in Xilen.

Wash 1x10 min in ethanol 100%.

Wash 1x10 min in ethanol 95%.

Wash 1x5 min in ethanol 70%.

Wash 2x5 min in TBS 1X.

Put the slides 10 min in endogenous peroxidase blocking solution: TBS 1x + 3% H2O2 + 10% methanol

Wash 3x5 min in buffer TBS 1X.

Put sections in 200 mL of citrate buffer 10mM, pH 6

Put sections in a boiling water bath for 20 min

Let sections cool down for 20 min at RT

Wash 3x5 min in TBS 1X.

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

Blocking in TBS 1X + 5% NGS (200uL/slides) 1h at RT

Gently wipe off the water ofthe slides

Put 200ul/slide of TBS 1X + 2% NGS + primary Ab48/72h (it depends of the Ab) at 4ºC (cold room)

Wash 3x5min in TBS 1X Buffer.

Gently wipe off the water ofthe slides

Put 200ul/slide TBS 1X + 2% NGS + Secondary Ab 1h at RT .

At this step it is important to prepare ABC solution and let it, at least, 30 min on the shaker

3x5min in TBS 1X

Incubate 1 hour at RT with ABC solution (4drops A/B in 10 mL of TBS 1x)

3x5min in TBS 1X

In aluminium foil: DAB Standard Kit: 1 drop of reagent B in 1 mL of reagent A (gives rise to brown staining)

In aluminium foil: Vector SG: 3 drops Chromogen + 3 drops Hydrogen Peroxide in 5mL PBS (gives rise to blue staining)

Cover tray plates with aluminium foil

Put 200 uL on each section and put a cardboard box on it to keep darkness for a time ranging from 3-15 minutes depending on the antibody used

Remove with an air-pump equipment and clean the material with bleach

3x5min in TBS 1X

1 min in ethanol 70%

1 min in ethanol 95%

1 min in ethanol 100%.

2x5 min in xylene

Puta line of mounting medium(DPX) byslide.Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles

Let dry the slides on the tray in the hood overnight