immunohistochemistryprotocol-Utexas.doc

IMMUNOHISTOCHEMISTRY PROTOCOL DAY 1 3 x Xylene deparaffinize @ RT (3 min each) 3 x 100% EtOH rehydrate @ RT (2 min each) 1 x 95% EtOH rehydrate @ RT (2 min) 1 x 80% EtOH rehydrate @ RT (2 min) 1 x 70% EtOH rehydrate @ RT (2 min) 1 x 1XPBS wash @ RT (5 min) Antigen retrieval 10 mM citric acid bufferpH3 @ 37oC (30 min) 3 x 1XPBS wash @ RT (5 min each) BLOCK ENDOGENOUS PEROXIDASES 1 x H2O2/MeOH soak @ RT (15 min) 90 ml methanol/10 ml 30%H2O2 3 x 1XPBS wash @ RT (5 min each) optional Pap pen circle sections @ RT shake off excess PBS prior to delimiting sections 1 x Blocking Buffer 1000 λ / slide @ RT (1 hr – over night) in a humidified chamber DAY 2 PRIMARY ANTIBODIES 1:750 10 ￥ options: DUAL FLUORESCENCE ( DILUTE incubate both “primary” TOGETHER ~1:50010 ￥ inMOUSE 1000 λ / slide @ RT (1 hr – over night) in a humidified chamber Blocking buffer ~1:50010￥ inGOAT 1000 λ / slide @ RT (1 hr – over night) in a humidified chamber Blocking buffer DAY 3 Drain 1 anti-body off section 3 x 1XPBS wash @ RT (10 min each) 1X PBS +0.1%-0.5%Tween-20 for some primary antibodies SECONDARY ANTIBODIES 1:200 20 ￥ options: DUAL FLUORESCENCE ( DILUTE incubate both “SECONDARY” TOGETHER 1:20020 ￥ * Cy-3 1000 λ / slide @ RT (5 min) in a humidified chamber RED: (goat α mouse) Blocking buffer α-MOUSE spin 5’ μ.fuge 2 remove ppt. 1:20020 ￥ * Cy-2 1000 λ / slide @ RT (5 min) in a humidified chamber GREEN : (goat α rabbit) Blocking buffer α-RABBIT spin 5’ μ.fuge 2 remove ppt. Drain 2 anti-body off section 5 x 1XPBS wash @ RT (10 min each) DAPI counter stain 1 x DAPI [10 μg/ml] 1000 λ / slide @ RT (10 min) in a humidified chamber 5 x 1XPBS wash @ RT (10 min each) MOUNT 1 x 70% EtOH dehydrate @ RT (1 dip) 1 x 80% EtOH dehydrate @ RT (1 dip) 1 x 100% EtOH dehydrate @ RT (1 dip) PermountTM ( mount coverslip Antigen retrieval solutions (10 mM CITRATE BUFFER PH3.0) 2.1 GRAMS CITRI