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Vt1000x vibratome

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The Leica VT1000X vibratome is a precision instrument used for sectioning biological samples. It employs a vibrating blade to create thin, uniform sections from fixed or embedded specimens. The VT1000X is capable of producing sections ranging from 10 to 500 micrometers in thickness, allowing for the preparation of high-quality samples for various microscopy and analysis techniques.

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Vibratome (897 citations)

6 protocols using vt1000x vibratome

1

Perfusion and Sectioning for Brain Immunohistochemistry

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The mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused through the left ventricle of the heart sequentially with ~5 ml heparinized saline followed by 30 ml of 3.75% acrolein and 2% paraformaldehyde in 0.1M phosphate buffer (PB; pH 7.4) (Milner, 2011 ). The brains were then removed and post-fixed in 2% acrolein and 2% paraformaldehyde in PB for 30 minutes. The brains were cut into 5-mm coronal blocks using a brain mold (Activational Systems Inc., Warren Michigan) and sectioned (40 μm thick) using a VT1000x Vibratome (Leica Microsystems, Buffalo Grove, IL). Sections were stored at −20°C in cyroprotectant solution (30% sucrose, 30% ethylene glycol in PB) until immunocytochemical processing (Milner, 2011 ).
Rubin B.R., Milner T.A., Pickel V.M., Coleman C.G., Marques-Lopes J., Van Kempen T.A., Kazim S.F., McEwen B.S., Gray J.D, & Pereira A.C. (2019). Sex and age differentially affect GABAergic neurons in the mouse prefrontal cortex and hippocampus following chronic intermittent hypoxia. Experimental neurology, 325, 113075.
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2

Brain Tissue Preparation and Sectioning

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Tissue was prepared using previously described procedures [22 (link)]. Mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and fixed by aortic arch perfusion, with 3–5 mL of saline (0.9%) containing 2% heparin followed by 30 mL of 3.75% acrolein and 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer pH 7.4 (PB). Brains were extracted from the skull and post-fixed in 1.9% acrolein and 2% PFA in PB for 30 min at room temperature. To ensure consistency in brain comparisons, brains were blocked coronally between the cauda hippocampus and pons using a brain mold (Activational Systems). Brains were coronally sectioned (40 μm thick) on a VT1000X vibratome (Leica Microsystems, Buffalo Grove, IL, USA) and stored at −20 °C in cryoprotectant (30% sucrose, 30% ethylene glycol in PB) until immunocytochemical processing.
Mazid S., Waters E.M., Lopez-Lee C., Poultan Kamakura R., Rubin B.R., Levin E.R., McEwen B.S, & Milner T.A. (2023). Both Nuclear and Membrane Estrogen Receptor Alpha Impact the Expression of Estrogen Receptors and Plasticity Markers in the Mouse Hypothalamus and Hippocampus. Biology, 12(4), 632.
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3

Pre-embedding Dual Immunolabeling Electron Microscopy

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Mouse brains were prepared and processed for pre-embedding dual immunolabeling electron microscopic studies according to previously described methods (Milner, 2011 (link)). For this, mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and their brains fixed by aortic arch perfusion consecutively with ~5 ml 2% heparin in normal (0.9%) saline followed by 30 ml of 3.75% acrolein and 2% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB; pH 7.4). After perfusion, the brains were removed from the skull and post-fixed in 2% acrolein and 2% PFA in PB at room temperature for 30 min. Brains were cut into 5-mm coronal blocks using a brain mold (Activational Systems, Inc., Warren, MI) and sectioned (40 μm thick) on a VT1000X Vibratome (Leica Microsystems, Buffalo Grove, IL). Brain sections were placed in cryoprotectant (30% sucrose, 30% ethylene glycol in PB) and then kept at −20°C until the immunocytochemistry experiment.
To ensure that sections from each experimental group were processed with identical immunolabeling conditions (Pierce et al., 1999 (link)), sections were marked in the cortex with punch codes and pooled into single containers. The region of the PVN (0.70-0.94 mm caudal to bregma (Hof et al., 2000 ) were chosen from two sections per animal (3 animals per group).
Ovalles A.C., Contoreggi N.H., Marques-Lopes J., Van Kempen T.A., Iadecola C., Waters E.M., Glass M.J, & Milner T.A. (2019). Plasma membrane affiliated AMPA GluA1 in estrogen receptor β-containing paraventricular hypothalamic neurons increases following hypertension in a mouse model of post-menopause. Neuroscience, 423, 192-205.
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4

Perfusion-fixation of Mouse Brains

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Mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, I.P.) and fixed by aortic arch perfusion, with 3-5 ml saline (0.9%) containing 2% heparin followed by either 30 ml of 4% PFA in 0.1M phosphate buffer (PB; sections for AR, PR and GPER1) or 30 ml 2% PFA and 3.75% acrolein in PB (light microscope sections for ERα and ERβ-EGFP and all sections for EM studies). Brains were extracted from the skull and post-fixed in either 4% PFA in PB (AR, PR and GPER1) at 4°C for 24 hours; or 2% PFA and 1.9% acrolein in PB (ERα and ERβ-EGFP) at room temperature for 30 min. Brains were coronally sectioned (40 μm thick) on a VT1000X vibratome (Leica Microsystems, Buffalo Grove, Ill., USA) and stored at −20°C in cryoprotectant (30% sucrose, 30% ethylene glycol in PB) until immunocytochemical processing (Milner, 2011 ).
Contoreggi N.H., Mazid S., Goldstein L.B., Park J., Ovalles A.C., Waters E.M., Glass M.J, & Milner T.A. (2021). Sex and age influence gonadal steroid hormone receptor distributions relative to estrogen receptor β-containing neurons in the mouse hypothalamic paraventricular nucleus. The Journal of comparative neurology, 529(9), 2283-2310.
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5

Perfusion-fixation and Brain Sectioning

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Three animals per group (males/females infused with AngII/saline) were deeply anesthetized with sodium pentobarbital as above and fixed by aortic arch perfusion sequentially with 2–3 ml saline (0.9%) containing 2% heparin followed by 30 ml of 3.75% acrolein and 2% PFA in PB (Milner et al., 2011 (link)). After the perfusion, brains were removed and post-fixed for 30 min in 2% acrolein and 2% PFA in PB at room temperature. Brains were then cut into 5 mm coronal blocks using a brain mold (Activational Systems, Inc., Warren, MI) and sectioned (40 μm thick) on a VT1000X Vibratome (Leica Microsystems, Buffalo Grove, IL). Brain sections were stored at −20°C in cryoprotectant until immunohistochemical processing (Milner et al., 2011 (link)).
To ensure identical labeling conditions between experimental groups for quantitative studies (Pierce et al., 1999 (link)), two sections per animal encompassing the region of the PVN [0.70–0.94 mm caudal to bregma; Fig. 1A (Hof et al., 2000)] were marked with identifying punches, pooled into single containers and then processed through all immunohistochemical procedures together.
Marques-Lopes J., Lynch M.K., Van Kempen T.A., Waters E.M., Wang G., Iadecola C., Pickel V.M, & Milner T.A. (2015). Female protection from slow-pressor effects of AngII involves prevention of ROS production independent of NMDA receptor trafficking in hypothalamic neurons expressing angiotensin-1A receptors. Synapse (New York, N.Y.), 69(3), 148-165.
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6

Electron Microscopy Analysis of Aging Mouse Brains

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The brains of mice from the peri-AOF (~4.5 months old) and post-AOF (~6.5 months old) time points were prepared for electron-microscopic studies [48 (link)]. For this, mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and fixed by aortic arch perfusion sequentially with 3–5 ml saline (0.9%) containing 2% heparin followed by 30 ml of 3.75% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer (PB) [48 (link)]. After the perfusion, brains were removed and postfixed for 30 min in 2% acrolein and 2% paraformaldehyde in PB at room temperature. Brains were then cut into 5-mm coronal blocks using a brain mold (Activational Systems Inc., Warren, Mich., USA) and sectioned (40 μm thick) on a VT1000X vibratome (Leica Microsystems, Buffalo Grove, Ill., USA). Brain sections were stored at –20°C in cryoprotectant (30% sucrose, 30% ethylene glycol in PB) until immunocytochemical processing.
To ensure identical labeling conditions between experimental groups for quantitative immunocytochemistry [60 (link)] , 2 sections per animal (3 animals per group) encompassing the region of the PVN (0.70–0.94 mm caudal to bregma; fig. 2a [61 ]) were marked with identifying punches, pooled into single containers and then processed together through all immunocytochemical procedures.
Marques-Lopes J., Tesfaye E., Israilov S., Van Kempen T.A., Wang G., Glass M.J., Pickel V.M., Iadecola C., Waters E.M, & Milner T.A. (2016). Redistribution of NMDA Receptors in Estrogen-Receptor-β-Containing Paraventricular Hypothalamic Neurons following Slow-Pressor Angiotensin II Hypertension in Female Mice with Accelerated Ovarian Failure. Neuroendocrinology, 104(3), 239-256.
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