Are you looking to isolate protein from the organ of corti?  You can dissect the bone off of the organ of corti and then isolate protein from there.  Otherwise, it would be helpful to know what exactly you are trying to do (western blot?)

f they are adult, removing the bonny capside is going to cause trauma and alter your results. I would place them in sterile PBS + protein inhibitor cocktail (+protease inhibitor cocktail if you are looking at phospho- proteins) and under sterile conditions smash them. Then pass through a 18 G needle, spinn down and collect the supernatant. If you want a membrane protein you will need to either sonicate or use a buffer with detergent like RIPA buffer or add SDS for example to your extraction buffer I prefer to sonicate than miracle buffers with secret recipes because it can interfere in your next experiment. Good luck

I have not heard of an issue of "trauma" or altered protein results from removal of the bony capsule surrounding the cochlea. If these effects are not of concern, using sharp instruments, dissection of the cochlea and removal of the stria vascularis and spiral ligament is easily done. The organ of Corti and other inner ear tissues will also be accessible. A sharp instrument (dental pick) can be used to make a pin-prick in the apex of the cochlea by slowly rocking or 'twirling' the point of the pick rather than pressing down the longitudinal access of the cochlea. Once a hole is made, the bone is chipped away by careful insertion of the dental pick and gentle outward motion of the pick. Working slowly (carefully), the cochlea should break apart fairly easily so that you can remove the soft tissue. Oftentimes, the soft tissue will remain and only the bony tissue is sloughed off, making removal of the soft tissue in one piece more likely. The stria vascularis and spiral ligament can be separated from each other with forceps. In my experience, if the animal is aged (>~4-6 months in the rat or mouse), it may require slightly more pressure to chip away the bone as it seems to have a harder consistency. Tissue can then be processed as for any soft tissue. If the cochlea is broken too harshly and bony spicules are attached, these tissues are likely to float away from large bone pieces. Alternatively, you might try passage through the 18Ga needle or a 25Ga needle to remove bony spicules as well.

Thanks everybody for nice suggestion, I am looking for isolation of cochlea for immunohistochemistry as well as western blotting. Please suggest for immunohistochemistry.

Pradip

For IHC is better that you decal 1st in 10%EDTA in a shaker for 1 week approx. Then dissect.

For the WB, I still think that since your tissue is not fixed if you try to remove the capside, that manipulation will introduce changes and can alter some proteins (phosphorylation, degradation, etc). But each one has their own protocol and after all your controls will undergo the same manipulation.

Thank you Dr. Bas for your kind suggestion and help

Dear Pradip, There s a very good description of the post-fix cochlear dissection on Charlie Liberman's website (along with a video): 

http://www.masseyeandear.org/research/otolaryngology/investigators/laboratories/eaton-peabody-laboratories/epl-histology-resources/cochlear-dissection-summary/

Please note this is for the mouse,  it is not a trivial dissection but it is do-able if you have good hands. The dissection in some other animals (e.g. guinea pig) is a lot easier. 

For immunocytochemistry it is also relatively easy to cryosection the fixed and de-calcified cochlea and then perform ICC on the sections (10-15µm). Of course you need to know your antigen is not affected by the de-calcification process. If your antibody will work in paraffin sections you can get better preservation of structure with those.

If you are dissecting un-fixed, non-decalcified tissue you have to be careful not to lose or damage the sensory epithelium (particularly if those are the cells you are interested in).

Finally for WB you will need a few  (the number depends on your efficiency) cochleae to get enough protein particularly if you are dissecting down to the part of the cochlea you are interested in.

Good luck!

For western blot, I have recently pooled 6 cochleae from 3 mice and i got ~ 4ug/ul protein. so its true that you might have to pool cochlea in order to get enough protein to see a band on a blot. All the steps have to be done on ice and quick so as to avoid degradation. I have extracted cochlea in 100 ul of RIPA buffer with protease and phosphatase inhibitor and then homogenized the bone and left on ice for 15 min. following this the homogenized tissue was centrifuged at 4 degree at maximum rpm for 15 min and you collect the supernatant. 

For IHC, fix the cochlea with intracardiac perfusion of 4% PFA followed by 1 hr post fixation in PFA. longer fixation can affect the antigenicity. fixed tissue is then decalcified  in EDTA for 2 days. this makes the tissue soft enough to dissect the organ of corti. you can then perform standard immunohistochemistry on the tissue.

I've also fixed by cardiac perfusion, but then followed by intra-cochlear perfusion by flushing

Thanks Dr kaur and Dr Curtis for your nice suggestion.

Pradip

Can anyone suggest for 4% PFA fixation time for P0 to P5 mice inner ear?