IHC Protocol (NBP1-02613)

Tissue Preparation: Formalin fixation and embedding in paraffin wax
Tissue Sectioning: Make 4-um sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60C.
Deparaffinization: Wash dry slides in 3 changes of xylene - 5 minutes each @ RT
Rehydration: Wash slides in 3 changes of 100% alcohol - 3 minutes each @ RT
Wash slides in 2 changes of 95% alcohol - 3 minutes each @ RT
Wash slides in 1 change of 80% alcohol - 3 minutes @ RT
Rinse slides in gentle running distilled water - 5 minutes @ RT
Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100C - 20 minutes
Remove from heat and let stand at room temperature in buffer - 20 minutes
Rinse in 1X TBS with Tween (TBST) - 1 minute @ RT
Immunostaining: (Do not allow tissues to dry at any time during the staining procedure)
Apply a universal protein block - 20 minutes @ RT
Drain protein block from slides, apply diluted primary antibody - 45 minutes @ RT
Rinse slides in 1X TBST - 1 minute @ RT
Apply a biotinylated anti-rabbit IgG (H+L) secondary - 30 minutes @ RT
Rinse slides in 1X TBST - 1 minute @ RT
Apply alkaline phosphatase streptavidin - 30 minutes @ RT
Rinse slides in 1X TBST - 1 minute @ RT
Apply alkaline phosphatase chromogen substrate - 30 minutes @ RT
Wash slides in distilled water - 1 minute @ RT
Dehydrate: (This method should only be used if the chromogen substrate is alcohol insoluble (e.g. Vector Red, DAB)
Wash slides in 2 changes of 80% alcohol - 1 minute each @ RT
Wash slides in 2 changes of 95% alcohol - 1 minute each @ RT
Wash slides in 3 changes of 100% alcohol - 1 minute each @ RT
Wash slides in 3 changes of xylene - 1 minute each @ RT
Apply coverslip