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      1. position:HOME > Support >
        Immunofluorescence
        Immunofluorescence: Common staining protocol 

        Paraffin sections 

        1- Deparaffinization / rehydration 
        - 30 min 50°C 
        - 3 x 3 min Xylol RT 
        - 3 min EtOH 96% 
        - 3 min EtOH 96% 
        - 3 min EtOH 80% 
        - 3 min EtOH 70% 
        - 30 min with PBS 

        2- Antigen Retrieval 
        - Autoclave: 10 min ,121°C 
         Buffer: Tris-HCL 10 mM, Tween 0,05%, pH9 

        (or alternatively use a microwave and heat it for 6 minutes without 
        cooking in order to avoid concentration changes) 

        - Let cool down in the buffer for 30 min 
        - 10 min PBS/Twin 
        - 10 min Glycin buffer (200ml dH2O + 4g Glycin) 
        - 10 min PBS/Twin 

        Immunofluorescence Staining 

        1- Encircle the tissue with Dako Pen 
        2- 30 min in PBS/Twin + 5% Goat serum (or serum from the same species as II ab) 
        (blocking) 


        Indirect Staining 

        3- 1h primary antibodies at RT (use shaking with rotation platform) 
        4- 2 x3 min with PBS/Twin 
        5- 30 min fluorochrom conjugated secondary antibodies at RT (use shaking). 

        From now on, please protect the slides from light!! 

        6- 2 x 3 min with PBS/Twin 

         Direct Staining 


        5- 1h direct conjugated antibodies at RT in PBS/Twin (shake) 

        6- 2 x 3 min with PBS/Twin 

        7- Counterstaining by use DAPI (1:100 000) in stabilize from DAKO, 5 min 
        8- 2 x 3 min with PBS/Twin 
        9- Cover slip slides by using a mounting medium that supports the endurance of 
        fluorescence (e.g. GelTol Aqueous Mounting Medium – Thermo/Shannon) 
        10- 20 min at RT (to harden the mounting medium) 

        11- Store at 4°C in darkness 

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