Pectoralis major tissue from five affected or non-affected chickens were cut at 2 mm2 and fixed with Karnovsky’s fixative in a weak vacuum for 2 h. Samples were rinsed three times with 0.05 M cacodylate buffer pH 7.2, post-fixed for 2 h in 1% osmium tetroxide, with 0.05 M cacodylate buffer. Samples were rinsed in distilled water and stained overnight in 0.5% uranyl acetate at 4.4°C. The tissues were dehydrated in a graded ethanol series then infiltrated with 50:50, Spurr’s medium: 100% ethanol for three changes. Samples were placed in fresh 100% Spurr’s medium, overnight with a weak vacuum. Fresh Spurr’s medium was pipetted into flat embedding molds, and tissues were placed in the molds and aligned. Molds were kept overnight in a weak vacuum. The molds were placed in a 70°C oven overnight. The tissues, in the cured blocks, were trimmed to 1 × 1 mm, and sectioned at 60–90 nm, using a diamond knife with an MT-2B ultra-microtome (Dupont Company, Newtown, CT). Sections were placed on 300 mesh copper grids and stained with 2% aqueous uranyl acetate, followed by lead citrate. Sections were viewed at 100 kV, with a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan).
Mt 2b ultramicrotome
Manufactured by DuPont
Sourced in United States
The MT-2B ultramicrotome is a laboratory instrument used for preparing thin sections of materials for microscopic analysis. It is designed to cut extremely thin samples, often in the range of nanometers, allowing for detailed examination of the microstructure of various materials.
Automatically generated - may contain errors
4 protocols using mt 2b ultramicrotome
1
Ultrastructural Analysis of Chicken Pectoralis
2
Ultrastructural Analysis of Granulysin-Induced Apoptosis
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Purified iRetics incubated with 100 nM GNLY ± 500 nM GzmB were
fixed in 2.5% buffered glutaraldehyde solution, 0.1 M, pH 7.2, for 3 hr
at 4°C, washed and the cell pellet was immersed in 4% agarose.
The pellet was fixed in 1% osmium tetroxide and 1.5% (w/v)
potassium ferrocyanide, dehydrated in ethanol and embedded in Araldite 502
(Electron Microscopy Sciences, Hatfield, PA, USA). Extra thin sections, obtained
using a Sorvall MT-2B ultramicrotome (Dupont, USA), were applied to 200-mesh
copper grids (Ted Pella, USA) and stained with 2% uranyl acetate and
Reynolds’ lead citrate. Images were obtained by transmission electron
microscopy using a Tecnai G2-12 - SpiritBiotwin FEI -120 kV (FEI, JP).
fixed in 2.5% buffered glutaraldehyde solution, 0.1 M, pH 7.2, for 3 hr
at 4°C, washed and the cell pellet was immersed in 4% agarose.
The pellet was fixed in 1% osmium tetroxide and 1.5% (w/v)
potassium ferrocyanide, dehydrated in ethanol and embedded in Araldite 502
(Electron Microscopy Sciences, Hatfield, PA, USA). Extra thin sections, obtained
using a Sorvall MT-2B ultramicrotome (Dupont, USA), were applied to 200-mesh
copper grids (Ted Pella, USA) and stained with 2% uranyl acetate and
Reynolds’ lead citrate. Images were obtained by transmission electron
microscopy using a Tecnai G2-12 - SpiritBiotwin FEI -120 kV (FEI, JP).
3
Ultrastructural Analysis of Granulysin-Induced Apoptosis
4
Transmission Electron Microscopy of Cells
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After FACS-sorting, cells were prepared as previously described [23] (link), [24] (link). Briefly, cells were fixed in 2.5% buffered glutaraldehyde solution, 0.1 M, pH 7.2, 6 h, 8°C. Cells were then washed with the same buffer. The pellets were included in phosphate buffer, 4% agarose and left overnight at 4°C. Next, the cells were fixed in a mixture of 1% osmium tetroxide and 1.5% (w/v) potassium ferrocyanide, dehydrated in a graded series of ethanol solutions, infiltrated, and embedded in Araldite 502 (Electron Microscopy Sciences, Hatfield, PA, USA). After polymerization, thin sections were obtained using a diamond knife on a Sorvall MT-2B ultramicrotome (Dupont, Wilmington, DE, USA) and mounted on uncoated 200-mesh copper grids (Ted Pella, Inc., Redding, CA, USA). Sections were stained with 2% uranyl acetate and Reynolds lead citrate and then analyzed using transmission electron microscopy (EM 10A Zeiss).
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