TITLE: Standardized immunohistochemical staining used in the Human Protein Atlas
AUTHORS: Anna Martinez Casals, Cecilia Lindskog

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. All IHC staining in the Human Protein Atlas project are performed using the following standard protocol. The primary antibody dilution is based on titration optimization, the dilution suggested by the Human Protein Atlas can be found under antibody and antigen information for each antibody. When primary antibody originates from other host animals than rabbit, there are some modifications and different secondary antibody is used.                </div></div>

[STEPS]
?. [Deparaffinization]
Dry paraffin sections at room temperature overnight.
?. [Deparaffinization]
Bake the paraffin sections  fromto  at .
?. [Deparaffinization]
Xylene incubation: incubate slides in xylene for .                                  incubate slides in xylene for .                                  incubate slides in xylene for .
?. [Deparaffinization]
96% ethanol incubation & 30%H2O2 (1:100): incubate slides for .
?. [Standard antigen retrieval method]
Heat the slides immersed in retrieval buffer for at in a pressure boiler (Decloaking chamber model DC2008INTL).
?. [Standard antigen retrieval method]
After completed boiling, leave the slides in the pressure boiler and let them cool down till .  Total time
?. [Immunohistochemical staining program, Autostainer 480]
Place slides in the Autostainer 480, all the incubations are done at room temperature.
?. [Immunohistochemical staining program, Autostainer 480]
Rinse slides in wash buffer.
?. [Immunohistochemical staining program, Autostainer 480]
Incubate slides with Ultra V Block for .
?. [Immunohistochemical staining program, Autostainer 480]
Rinse slides in wash buffer.
[once more, total washes 2]
?. [Immunohistochemical staining program, Autostainer 480]
Incubate slides with primary antibody, diluted in antibody Diluent OP, for .
?. [Immunohistochemical staining program, Autostainer 480]
Rinse slides in wash buffer .
[two times more, total washes 3]
?. [Immunohistochemical staining program, Autostainer 480]
Incubate slides with labeled HRP polymer for .
?. [Immunohistochemical staining program, Autostainer 480]
Rinse slides in wash buffer .
[once more, total washes 2]
?. [Immunohistochemical staining program, Autostainer 480]
Incubate slides with DAB solution for .
?. [Immunohistochemical staining program, Autostainer 480]
Rinse slides in distilled water.
?. [Counterstaining and coversliping]
Transfer slides in the Autostainer XL.
?. Counterstain slides with hematoxylin for .
?. Rinse slides in lithium carbonate water diluted 1:5 from saturated solution for .
?. Rinse slides in tap water for .
?. 80% ethanol incubation: incubate slides in 80% ethanol for .
?. Mount coverslip in each of the slide using Pertex as a mounting media.
?. [Deparaffinization]
Ethanol absolute incubation: incubate slides in ethanol absolute for .                                                    incubate slides in ethanol absolute for .
?. [Deparaffinization]
96% ethanol incubation: incubate slides in 96% ethanol incubation for .
?. [Deparaffinization]
80% ethanol incubation: incubate slides in 80% ethanol incubation for .
?. [Deparaffinization]
Distilled water: : incubate slides in distilled water or until antigen retrieval step.
?. 96% ethanol incubation: incubate slides in 96% ethanol for.                                           incubate slides in 96% ethanol for.
?. 99% ethanol incubation: incubate slides in 99% ethanol for.                                           incubate slides in 99% ethanol for.                                           incubate slides in 99% ethanol for.
?. NeoClear incubation: incubate slides in NeoClear for.                                      incubate slides in NeoClear for.