Table of Contents
Adapted from the Allen Institute for Neural Dynamics SOP
1.1 - Select a plastic well plate for free-floating tissue section staining. We use either a 6-well plate or a 24-well plate. For 24-well plates, you can often fit 8-10 slices/well (and uses about 500uL of buffer for washes or stains or blocking).
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NOTE: A plastic well plate may be helpful for conducting multiple different stains simultaneously, while a Netwell kit may be efficient for applying the same stain to a large number of tissue sections.
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1.2 - Fill wells with 1xPBS and place tissue sections to be stained into wells.
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NOTE: If not moving into staining right away, tissue can be placed into 1xPBS + 0.05% Sodium Azide solution in the wells for about a week (although the ECM is best labeled within 2 days).
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NOTE: If there are a large amount of tissue sections, the sections may be double or triple stacked in each well in the well plate (or more if using Netwell insert), but ideally sections in the same well should be separated by at least a few hundred microns (ex: every 6th section or more) so it is easier to see anatomical differences and determine section order when mounting.
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1.3 - If tissues will be stained in different conditions (ex: staining sections with different primary or secondary antibodies) it is best to draw a map of these conditions on the plastic well plate lid and a corresponding map in lab notebook.
(II – IX Staining)
2.1 – Once sectioned, wash mouse brain sections three times in 1xPBS for 00:05:00 at Room temperature on shaker.
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NOTE: In this step and all subsequent steps, protect tissue from light to prevent bleaching by wrapping well plate in foil while it is on the shaker. This is less important prior to applying fluorescent tags, but a good habit.
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3.1 - Select blocking solution. Depends on the staining and antibodies used.