Animals were killed and excised inner ears were fixed overnight in 2.5% gluteraldehyde in 0.1 M phosphate buffer (Sigma-Aldrich). Fixed ears were decalcified for 48 h in 4.3% EDTA in 0.1 M phosphate buffer (Sigma-Aldrich). Fine dissection was performed to reveal the organ of Corti, before osmium tetroxide (Agar Scientific)—thiocarbohydrazide (Fluka) (OTOTO) processing (adapted from ref. 38 (link)) was carried out. The inner ears were then dehydrated through increasing strength ethanol solutions (Fisher Scientific) and critical point dried using an Emitech K850 (EM Technologies LTD). The specimens were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150T sputter coater (Quorum Technologies). Prepared cochleae were visualised with a JEOL LSM-6010 (Jeol Ltd.) scanning electron microscope. Hair cell counts were performed by counting the number of OHCs adjacent to 10 pillar cells, for the analysis the cochlea was divided into three separate regions (turns), apical (<90° from apex), mid (180–360° from apex) and mid-basal (360–540° from apex). Ears from at least three mice were analysed for each genotype at each turn and time point.
Lsm 6010
Manufactured by JEOL
The JEOL LSM-6010 is a scanning electron microscope (SEM) designed for high-resolution imaging and analysis of samples. It features a tungsten filament electron source, secondary electron and backscattered electron detectors, and a high-vacuum system. The LSM-6010 is capable of producing magnified images of sample surfaces with a resolution up to 3 nanometers.
Automatically generated - may contain errors
Lab products found in correlation
Silver paint, by Agar Scientific (4 mentions)
Increasing strength ethanol solutions, by Thermo Fisher Scientific (3 mentions)
Quorum q150t sputter coater, by Quorum Technologies (3 mentions)
0.1 m phosphate buffer, by Merck Group (1 mentions)
Phosphate buffer, by Merck Group (1 mentions)
Osmium tetroxide, by Agar Scientific (1 mentions)
Quorum q150r s sputter coater, by Quorum Technologies (1 mentions)
Ht7700, by Hitachi (1 mentions)
4 protocols using lsm 6010
1
Scanning Electron Microscopy of Mouse Cochlea
2
Scanning Electron Microscopy of Organ of Corti
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Animals were euthanised and excised inner ears were fixed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer (Sigma-Aldrich), then decalcified for 48 h in 4.3% EDTA in 0.1 M phosphate buffer (Sigma-Aldrich). Fine dissection was performed to reveal the organ of Corti, before osmium tetroxide (Agar Scientific)-thiocarbohydrazide (Fluka) (OTOTO) processing (adapted from Hunter-Duvar, 1978 (link)) was carried out. Samples were then dehydrated through increasing-strength ethanol solutions (Fisher Scientific) and critical point dried using an Emitech K850 (EM Technologies Ltd). Specimens were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150T sputter coater (Quorum Technologies). Prepared cochleae were visualised with a JEOL LSM-6010 (Jeol Ltd) scanning electron microscope. Hair-cell counts were performed by counting the number of adjacent IHCs and OHCs to 20 pillar cells; for the analysis, the cochlea was divided into four separate regions (turns): apical (<90° from apex), mid-apical (90-180° from apex), mid (180-360° from apex) and mid-basal (360-540° from apex). Ears from at least three mice were analysed for each genotype at each turn and time point.
3
Ultrastructural Analysis of Mouse Cochlea
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Animals were euthanized and excised inner ears were fixed overnight in 2.5 % gluteraldehyde in 0.1 M phosphate buffer (Sigma-Aldrich). Fixed ears were decalcified for 48 hours in 4.3 % EDTA in 0.1 M phosphate buffer (Sigma-Aldrich). Fine dissection was performed to reveal the organ of Corti, before osmium tetroxide (Agar Scientific)–thiocarbohydrazide (Fluka) processing (adapted from Hunter-Duvar [18 (link)]) was carried out. The inner ears were then dehydrated through increasing strength ethanol solutions (Fisher Scientific) and critical point dried using an Emitech K850 (EM Technologies Ltd). The specimens were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150R S sputter coater (Quorum Technologies). Prepared cochlea were visualised with a JEOL LSM-6010 (Jeol Ltd) scanning electron microscope. Hair cell stereocilia bundle counts were performed by counting the number of adjacent inner hair cells (IHCs) and outer hair cells (OHCs) to ten pillar cells. For the analysis the cochlea was divided into three separate regions (turns), apical (<180° from apex), mid (180–450° from apex), and basal (450–630° from apex). Four ears (one ear per mouse) were analysed for each genotype at each region.
4
Multimodal Kidney Tissue Preparation
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For light microscopy, kidneys fixed in 10% neutral-buffered formalin were embedded in paraffin wax and sectioned at 5 μm. Kidney sections were stained with hematoxylin and eosin, periodic acid–Schiff, and Masson trichrome stain.
For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), 1-mm3 cubes of kidney cortex were fixed in 3% glutaraldehyde and 4% formaldehyde in 0.1 M PIPES (the common name for piperazine-N,N′-bis(2-ethanesulfonic acid) buffer, pH 7.2 (minimum, 1 hour).
For TEM, specimens were then rinsed in 0.1 M PIPES buffer, postfixed in 1% buffered osmium tetroxide, rinsed in buffer, block stained in 2% aqueous uranyl acetate, dehydrated in an ethanol series, and embedded in TAAB resin (TAAB Laboratories). Gold–silver sections were cut, stained with Reynolds lead stain, and viewed on a Hitachi HT7700 transmission electron microscope.
For SEM, samples were then dehydrated through increasing strength of ethanol solutions and critical point dried using an Emitech K850 (EM Technologies LTD). Three specimens per animal were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150T sputter coater (Quorum Technologies). The specimens were untimely visualized with a JEOL LSM-6010 (Jeol Ltd.).
For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), 1-mm3 cubes of kidney cortex were fixed in 3% glutaraldehyde and 4% formaldehyde in 0.1 M PIPES (the common name for piperazine-N,N′-bis(2-ethanesulfonic acid) buffer, pH 7.2 (minimum, 1 hour).
For TEM, specimens were then rinsed in 0.1 M PIPES buffer, postfixed in 1% buffered osmium tetroxide, rinsed in buffer, block stained in 2% aqueous uranyl acetate, dehydrated in an ethanol series, and embedded in TAAB resin (TAAB Laboratories). Gold–silver sections were cut, stained with Reynolds lead stain, and viewed on a Hitachi HT7700 transmission electron microscope.
For SEM, samples were then dehydrated through increasing strength of ethanol solutions and critical point dried using an Emitech K850 (EM Technologies LTD). Three specimens per animal were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150T sputter coater (Quorum Technologies). The specimens were untimely visualized with a JEOL LSM-6010 (Jeol Ltd.).
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