Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Inner ear extraction and mild fixation (steps 1–15) (A) Hemi-section of a P11 mouse head with removed skin (steps 1–2). (B and C) The brain is removed with a spatula, and the portion of the temporal part of the skull containing the inner ear is cut out and placed in a 35 mm dish with dissection buffer (steps 2–4). The blood vessels surrounding the inner ear can be used as a cutting landmark. Graphics of the inner eare adapted with permission from Shutterstock. (D and E) The inner ear is detached from the skull (steps 5–6). (F) Using a spoon, the inner ear is transferred to a 35 mm dish containing mild fixative, and the mesenchymal tissue and stapes ossicle are removed (step 7–9). (G) Make a hole at the cochlear apex (step 10). (H) Using a 1 mL syringe and a 35G needle, perfuse 0.5 mL of the mild fixative through the oval window. The fixative should be observed exiting the apical hole (steps 11–13). (I) Repeat the step through the round window (step 14). (J) Incubate in the fixative for 30 min without shaking and at RT (step 15).

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Extraction, Dissection

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Organ of Corti dissection (steps 16–24) (A) After fixation, the inner ear is transferred to a dish containing dissection buffer (step 16). (B and B') A cartoon representing a cross-section of a cochlear turn before its dissection and picture of a P7 mouse inner ear before dissection (step 17). (C and C') Cochlear bone is removed (step 18). (D and D′) Stria vascularis and inner sulcus are removed, pinching the structures at the base of the cochlea and unrolling them toward the apex (step 19). (E and E') The Reissner's membrane is removed (step 20). (F and G) The translucent tectorial membrane covers the hair cell hair bundles and is removed from the organ of Corti (step 21). (H) The organ of Corti is free from the tectorial membrane and still attached to the central cochlear bone, which facilitates downstream sample manipulations (step 22). (I) The dissected cochlea is transferred to a 2 mL round bottom tube containing strong fixative. The tube is kept a 4°C (without agitation) for at least 2 h (step 23–24).

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Dissection, Membrane

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Sample dehydration and critical point drying (steps 25–34) (A) After fixation, the samples are washed with dissection buffer (2 × 5 min) and then washed with ice-cold H 2 O, 15%, 30%, 50%, 75%, 95%, and twice 100% ethanol for 5 min each and on ice (steps 25–27). (B) The samples are transferred with a spoon to a critical point drying sample holder that has itself been submerged in a glass container filled with ice-cold 100% ethanol (steps 28–29). (C and D) The critical point drying sample holder is placed in the chamber of a critical point drying instrument, pre-filled with 100% ethanol. The instrument will cool the ethanol, replace it with liquid CO 2 , and reach the CO 2 critical point drying at 31°C and 1,070 psi. At this point, liquid CO 2 will come fluid and slowly effuse from the sample without damaging the sample structure (steps 30–33). (E) After the procedure, the samples become white and highly fragile. The samples can be stored in a desiccator or directly mounted. The diameter of a well from this sample holder (2 × 12 wells) is 4 mm (step 34).

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Dissection

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Dried-sample dissection, mounting on a stub, and metal coating (steps 35–46) (A) The sample must be further dissected to minimize the distance between the tissue of interest and the metallic mounting stub. Because the samples are highly sensitive to electrostatic charges and can “fly” away easily, a sample is temporarily held in place on a double-sided carbon tape placed on a stub (a so-called holder stub) (steps 35–37). (B) Dried apical turn of a P11 mouse cochlea (steps 35–37). (C) A mixture of silver paint and epoxy is placed on another stub (the mounting stub). This stub has a 45° radius beveled face that is optimal for imaging the front side of hair bundles (steps 38–39). (D) Parts of the cochlear turn are positioned tangentially to the stud middle edge (step 40). (E) After drying 15–20 h in a desiccator, a thin layer of metal is deposited onto the sample. To coat all sides of the hair bundles and obtain optimal conductivity, the coating is first performed on the backside (using a custom-made adaptor) with 2 nm of Palladium (step 45a). (F) Next, the front side of the hair bundles are coated with 2 nm of Palladium (steps 45b). (G) A total of 4 nm of palladium had been coated onto the hair bundle with a sputter coater instrument (step 46). (H) An uncoated sample for visual comparison with the coated sample in G (step 46). Stub diameter: 11 mm, Scale bar: B: 0.5 mm

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Dissection, Imaging, Comparison

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet:

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Recombinant, Electron Microscopy, Dissection, Microscopy

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Dissection buffer

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Dissection, Concentration Assay

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Mild fixative

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Concentration Assay, Dissection

Journal: STAR Protocols

Article Title: High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

doi: 10.1016/j.xpro.2022.101213

Figure Lengend Snippet: Strong fixative

Article Snippet: Dissection scissors , Fine Science Tools , Cat#14090-09.

Techniques: Concentration Assay, Dissection