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    • CommentAuthorkaswart
    • CommentTimeMay 26th 2010
    1.) Thaw sections at room temperature for 5 minutes.
    2.) Incubate the slides in PBS + 0.1% Triton for 30 minutes to 1 hour to permeablize tissue.
    3.) Rinse 3 times for 5 mins in PBS.
    4.) Block by overlaying slides with 1 ml PBS + 2% HINGS for 1 hour at room temperature (RT).
    **NOTE: Other blocking serums--such as goat or sheep--may be used instead of HINGS.
    5.) Remove block and replace with primary antibody diluted in PBS + 2% HINGS. Cover each slide with a piece of parafilm to prevent evaporation.
    6.) Incubate overnight in cold room OR for a few hours at RT.
    7.) Rinse slides 3 times for 10 mins in PBS.
    8.) Add secondary antibody diluted in PBS + 2% HINGS and incubate 1 hour.
    **NOTE: Fluorophore-conjugated secondary antibodies will bleach if exposed to light. Always keep antibody stock and slides away from light to protect the fluorophore.
    9.) Wash 3 times for 10 mins in PBS in the dark; add 80 ul Fluoromount and coverslip. Remember to store slides awat from light to reduce bleaching.
    • CommentAuthorjakhan66
    • CommentTimeMay 16th 2013

    Refer to Day 3 (steps 13-18) and Day 4 in the link provided below for detailed modfication to this protocol: