TITLE: General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections
AUTHORS: Tereza Blassova

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Deparaffinize slides in 3 changes of xylene, 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Drain off the blocking buffer from the slides.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Block endogenous peroxidase activity by incubating sections in Peroxidase-Blocking solution (Dako) at room temperature for 10 min to block endogenous peroxidase activity.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Rinse in 300ml of PBS for 3 changes, 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Optional: Perform antigen retrieval to unmask the antigenic epitope. The most commonly used antigen retrieval is a citrate buffer method. Arrange the slides in a staining container. Pour 300ml of 10mM citrate buffer, pH 6.0 into the staining container and incubate it at 95-100°C for 10 min (optimal incubation time should be determined by user). Remove the staining container to room temperature and allow the slides to cool for 20 min.  We used different ways depend to used primary antibody: - Heat-induced epitope retrieval in Epitope Retrieval Solution pH 9 X pH 6 (Novocastra Leica, Leica Biosystems GmbH, Nussloch, Germany); 20 minutes                 - Proteinase K (Dako) - 10 min.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Rinse slides in 300ml PBS for 3 changes, 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Optional: Add 200µl blocking buffer (e.g. 10% goat serum in PBS) onto the sections of the slides and incubate in a humidified chamber at room temperature for 1 hour.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Apply 200µl appropriately diluted primary antibody (in antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature overnight.
[overnight]
4 °C
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Apply 200µl appropriately diluted secondary antibody (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.We used: N-Histofine® Simple Stain MAX PO (NICHIREI CORPORATION INC.)Apply  2  drops  of  Simple  Stain  MAX  PO  (MULTI)  to  each  slide  so as to  provide  a  complete  cover  of  the  sections.  Incubate  at  room  temperature  for  30 minutes.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Wash slides in 300ml PBS for 3 changes, 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Apply 150 µl DAB Liquid solution (Dako) to the sections on the slides to reveal the color of antibody staining. Allow the color development for
(Caution: DAB is a suspect carcinogen. Handle with care. Wear gloves, lab coat and eye protection.)
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Rinse slides in destil water, 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Optional: Counterstain slides by immersing sides in Hematoxylin (e.g. Gill's Hematoxylin) for 1-2 min.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Rinse the slides in running tap water for > 15 min.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Clear the tissue slides in 3 changes of xylene and coverslip using mounting solution. The mounted slides can be stored at room temperature permanently.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Observe the color of the antibody staining in the tissue sections under microscopy.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Wash the slides in 300ml PBS for 3 changes 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Dehydrate the tissue slides through 4 changes of alcohol (95%, 95%, 100% and 100%), 5 min each.
?. [General Immunohistochemical Protocol for formalin-fixed, paraffin-embedded tissue sections]
Transfer slides to 100% alcohol, for 2 changes, 3 min each, and then transfer once through 95%, 70% and 50% alcohols respectively for 3 min each.