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    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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