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Dry the sections o/n (or at least 1 hour) at room temperature.
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Put the slides into the Coplin jar for washes
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Wash 3-6 x 3 minutes in TBS-T (to dissolve the OCT)
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Draw circles around the tissue with a hydrophobic pen or just dry the slide around the tissue before each incubation step to provide surface tension
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If your tissue was fresh frozen, fix the tissue for 10 minutes in 75% acetone + 25% ethanol or 4% PFA -> Wash 3-6 x 3 minutes in TBS-T
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If using tyramide signal amplification (TSA): Add approx. 100 μl 1 % peroxide (dilute in MQ water) and wait until you see bubbles (3 minutes) -> Wash 3 x 3 minutes in TBS-T
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If using primary labelled or primary followed by secondary antibody, skip peroxide step.
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Block non-specific binding, generally related to isotype. General block is casein/Background sniper (for specific antibodies, the protein found in milk powder). Can use undiluted or anywhere from 1:10 to 1:100 depending on your antibody
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Dilute primary antibody in appropriate diluent (pH of diluent is antibody specific, read the datasheet: primary antibody dilution is determined empirically through testing and is different for every antibody: use approx. 200 μl / sample)
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Incubate for 30-120 minutes in the wet chamber at room temp, overnight at 4 degrees
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Wash 6 x 3 minutes in TBS-T
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If using primary fluorescently labelled antibody, skip to 15
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if using secondary fluorescently conjugated antibody, use 1:500 dilution of secondary antibody
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If using TSA, detect the primary antibody by using the correct Anti-XXX IgG
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ImmPRESS HRP Anti-XXX IgG (Peroxidase) Polymer Detection Kit, Vector Laboratories:
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Wash 6 x 3 minutes in TBS-T
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TSA flurophore of interest (Perkin Elmer):
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Dilute 1:50 in the provided diluent (use approx. 100 μl / sample; you can increase the dilution even further, but the diluent is the limiting factor)
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Incubate for 7-10 minutes in the wet chamber (covered!)
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Wash 6 x 3 minutes in TBS-T
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Add DAPI
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Wash 2 x 3 minutes in TBS-T
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Wash 1 x in MQ water to remove debris from slide, dry around tissue.
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Wash coverslip once with MQ water to remove debris from coverslip.
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Add a drop of mounting media onto the tissue. Place dry coverslip onto mounting media. Do not create bubbles between the slide and coverslip, if bubbles occur, push them out the side.
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Seal the slide with a sealant or let the mounting media cure for at least 60 minutes.
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Cleaning slide: Always clean both slide and coverslip.
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The best images are collected directly after staining. But slides stained like this can be stored short-term at 4 °C, or frozen at -20 °C and stored for later analysis.