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    Protocols
    Bioss is dedicated to helping you achieve exceptional results. Our top-notch scientific support team has worked hard to develop these protocols for all our applications. We hope these instructional aids assist you in your research!







    Immunohistochemistry Protocol for Frozen Tissuepdf iconDOWNLOAD A PDF

    This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different tissue samples.

    Tissue Preparation

    Note: For tissue stored at -80°C, remove from freezer and equilibrate at -20°C for about 15 minutes before sectioning. This prevents cracking of the block during sectioning.

    1. Cut sections at 4-8 μm and place on pre-cleaned and positively charged microscope slides.
    2. Air dry sections on bench for a few minutes to help the sections adhere to the slides.
    3. Fix sections in precooled acetone for 10 minutes at 4°C.
    4. Wash with Tris-Buffered Saline (TBS) to remove all traces of acetone.
    Immunostaining

    Recommended: Do not allow tissues to dry at any time during the staining procedure.

    1. Rinse slides 2 times with Tris-Buffered Saline + Tween (TBST) for 5 minutes each at RT.
    2. Block with 5% serum or BSA for 2 hours at RT.
    3. Drain blocking buffer from slide.
    4. Incubate slides with the diluted primary antibody overnight at 4°C with gentle agitation.
    5. Wash slides 2 times with TBST for 5 minutes at RT.
    6. Incubate slides with diluted conjugated secondary antibody for 2 hours at RT with gentle agitation.
    7. Wash slides 2 times with TBST for 5 minutes at RT.
    8. Develop with chromogen for 10 minutes at RT.
    9. Wash slides in distilled water for 1 minute at RT.
    10. Counterstain (if required).
    11. Mount coverslips.

     



    Immunohistochemistry Protocol for Paraffin Embedded Tissue pdf iconDOWNLOAD A PDF

    This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different tissue samples.

    Tissue Preparation (Formalin Fixed, Paraffin-embedded Sections)
    1. Cut sections at 4um and place on pre-cleaned and positively charged microscope slides.
    2. Heat in a tissue-drying oven for 45 minutes at 60°C.
    Deparaffinization and Rehydration
    1. Wash slides 2 times in Xylene for 3 minutes each time at room temperature (RT).
    2. Wash slides in Xylene 1:1 with 100% ethanol for 3 minutes at RT.
    3. Wash slides 2 times in 100% ethanol for 3 minutes each at RT.
    4. Wash slides 2 times in 95% ethanol for 3 minutes each at RT.
    5. Wash slides in 70% ethanol for 3 minutes at RT.
    6. Wash slides in 50% ethanol for 3 minutes at RT.
    7. Rinse slides gently with running distilled water for 5 minutes at RT.
    Antigen Retrieval
    1. Boil slides in 0.01M sodium citrate buffer (pH6) at 100°C for 15-20 minutes. Remove the slides from heat and allow them to stand at RT in buffer for 20 minutes.
    2. Rinse twice with Tris-Buffered Saline + Tween (TBST) for 5 minutes at RT.
    Immunostaining

    Recommended: Do not allow tissues to dry at any time during the staining procedure.

    1. Block endogenous peroxidase with 3% hydrogen peroxide for 30 minutes.
    2. Block with 5% serum or BSA for 2 hours at RT.
    3. Drain blocking buffer from slide.
    4. Incubate slides with the diluted primary antibody overnight at 4°C with gentle agitation.
    5. Wash slides 2 times with TBST for 5 minutes at RT.
    6. Develop with chromogen for 10 minutes at RT.
    7. Wash slides in distilled water for 1 minute at RT.
    8. Counterstain (if required).
    9. Dehydrate when using a chromogen substrate that is alcohol insoluble by washing slides in 80%, 95%, 100% and Xylene each for 1 minute at RT.
    10. Mount coverslips.