Immunostaining, hair cell and synaptic counts

Whole mount immunostaining for ribbon synapse

(CtBP2 – GluR2 – MyosinVIIa triple label for mouse)

- Perfuse the temporal bone with 4% paraformaldehyde, post-fix overnight on shaker at 4°C

- Decalcify in EDTA, 2-3 days

- Micro-dissection of utricle/saccule and cochlear sensory epithelia (remove nerve stubs so that the sensory epithelia can lay flat)

- Incubate the sample pieces in 30% Sucrose in 8um pore-size transwell insert and dish, shake at room temperature for 15 min. Make sure all pieces sink to the bottom.

- Freeze the samples in transwell insert and dish at -80C, thaw at room temperature, rinse with PBS/0.3% triton X-100. (This step helps with permeabilization)

- Blocking: 5% NHS in PBS/0.3% triton, one hour on shaker at room temperature

- Primary antibodies, overnight in 37°C oven (or room temperature depending on antibodies)

mouse(IgG1) anti-CtBP2 @ 1:200 (1:100 with glycerol)             BD Transduction Labs, #612044

mouse(IgG2a) anti-GluR2 @ 1:2000 (1:1000 with glycerol)    Millipore, #MAB397

rabbit anti-MyosinVIIa @ 1:200                       Proteus BioSciences, #25-6790

5% NHS with 0.3% triton

- Rinse (PBS/0.3% triton, 3X, 15 min total)

- Secondary antibodies, 1-2 hours @ 37° C

goat anti-mouse(IgG1)-AF568 @ 1:500                  Invitrogen, #A21124

goat anti-mouse(IgG2a)-AF488 @ 1:500                Invitrogen, #A21131

donkey anti-rabbit-AF647 @ 1:200                         Invitrogen, #A31573

5% NHS with 0.3% triton

- Rinse (PBS/0.3% triton, 3X, 15 min total)

- Mount and coverslip in VectaShield                 Vector Labs, #H-1000

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