[Histonet] BRDu IHC protocol

[Galina Deyneko]

 Hi Luis
I attach my protocol for BRDu
Best regards
BRDU protocol
1.            De-wax
and hydrate through the changes of Xylenes and graded Ethanols (100%, 95%, 70%)
the paraffin slides till DI water.
2.            HIER
:Performed in Biocare’s Decloaking Chamber with manufacture settings ( set
point 1-125˚C, 30 seconds, set point 2- 90˚C , 10 seconds) in plastic container
with 1X Rodent Decloaker solution which is stable modified Citrate Buffer pH
6.00, (Biocare # RD913).
Optional:
     Place the slides
in preheated till 90- 100 ˚C in 1X Rodent Decloaker solution which is stable
modified Citrate Buffer pH 6.00, (Biocare # RD913) and steam 45 minutes in
steamer .Remove the slides from steamer and allow them to cool till RT˚
3.            Rinse in
DI water and Wash in Tris buffer three times x 5 minutes
4.            Circle
the tissue on the slides with hydrophobic barrier using PAP pen
5.            Incubate
with pepsin enzyme digestion solution – 2 minutes. 
6.            Prepare
fresh before using: 0.25%  solution of
pepsin (250 mg of powder in 100 ml of Tris) (powder, Biomedicals, # 195367 )
in  1X Tris buffer( 10X Fisher
Scientific, BP2471), titrated to pH 2.00 with concentrated hydrochloric acid.
Optional: Preparation of 0.05M Tris(which correspond to 1X
Tris) from 1M Tris (Teknova. ,cat.# T1068,pH6.8)- 10ml of 1 M Tris + 190 ml of
DI water. (53 mg of powdered pepsin in 22.200 ml of Tris, pH2.5) 
7.            Neutralize
the slides 2 times x 5 minutes each in the 0,1 M Borate Buffer , pH 8.5 (0.5M
sodium Borate Buffer, Boston Bioproducts, # BB-66, 1 part of 0.5M buffer + 4
parts of DI water).
8.            Wash in
Tris buffer three times x 5 minutes.
9.            Quench
the endogenous peroxidase with PEROXIDAZED 1 (Biocare # PX 968)  for 15 minutes at RT 
10.          Rinse in
DI water and Wash in Tris buffer two times x 5 minutes
11.           Block
endogenous Protein  with Background
Sniper for 30 minutes at RT ( Biocare , #BS966)
12.          Tap off
the excess of protein block , do not rinse
13.          Incubate
the slides with the primary antibody (mouse
monoclonal anti- bromodeoxyuridine, Roche, # 11 170 376001) 80
minutes. Diluent: ready-to-use antibody diluent, (Dako, # S0809).
Dilutions:  1:700 Wash in Tris buffer
three  times x 5 minutes
14.          Incubate
the slides with mouse –on-mouse HRP Polymer (Biocare, #MM620) for 30 minutes at
RT.-for mouse tissues or with mouse on rat HRP polymer (Biocare, # MRT 621) for
rat tissues.
15.          Wash in
Tris buffer two times x 5 minutes
16.          Stain the
slides with freshly prepared DAB chromogen (Dako, #K3468) and developed
end-colored product in the targeted cells under microscope. Staining is
complete when the protein positive areas turn to dark brown and protein
negative areas (background) remain colorless. Stop the chromogen reaction by
placing the slides in DI water with following rinse in fresh DI water to remove
the residual DAB and clear the slides.- 5 minutes.

Galina Deyneko
Novartis, Cambridge, MA
 
617-871-7613 w