Mouse spinal cords were fixed in 2.5% glutaraldehyde and 4% PFA in 0.1 M sodium cacodylate buffer at pH 7.4 after deep anesthesia perfusion. Spinal cords were vibratome-sectioned and immersion fixed in the same buffer for 24 h at 4°C. After tissue trimming and washes in 0.1 M sodium cacodylate buffer, postfixation in reduced osmium (2% osmium, 2.5% potassium ferrocyanide in 0.1 M cacodylate buffer) was followed by en bloc uranyl acetate (1% aqueous uranyl acetate) contrasting, graded dehydration in ethanol, and embedding in epon resin (Serva). After ultrathin sectioning, the grids (UC7 Ultramicrotome; Leica) were contrasted by 1% uranyl acetate and lead citrate (Ultrostain; Leica). Semithin sections were contrasted by an equimolar mixture containing 1% methylene blue (Carl Roth GmbH & Co. Kg) in 100 ml sodium tetraborate and 1% (1 g) azure Blue (Carl Roth) in 100 ml water.
Ultrostain
Manufactured by Leica
Sourced in Germany
Ultrostain is a high-performance staining system designed for use in clinical and research laboratories. It is capable of automated staining of tissue sections, blood smears, and cytology samples. The Ultrostain system provides consistent and reproducible staining results, ensuring reliable and efficient sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Uc7 ultramicrotome, by Leica (3 mentions)
Epon resin, by Serva Electrophoresis (3 mentions)
Jem 1400 plus tem, by JEOL (2 mentions)
Ruby 8 mpx ccd camera, by JEOL (2 mentions)
Azure blue, by Carl Roth (1 mentions)
Methylene blue, by Carl Roth (1 mentions)
Em uc6, by Leica (1 mentions)
H 7100, by Hitachi (1 mentions)
Jem 1400plus, by JEOL (1 mentions)
Potassium ferrocyanide, by Merck Group (1 mentions)
Uranyl acetate, by Leica (1 mentions)
Osmium tetroxide, by Science Services (1 mentions)
Em grade, by Science Services (1 mentions)
5 protocols using ultrostain
1
Ultrastructural Analysis of Mouse Spinal Cords
2
Ultrastructural Analysis of PV-DAB Synapses
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PV-DAB immunolabeled sections were prepared for qualitative electron microscopic analysis. Areas of interest in layer III were re-embedded and 60 nm ultrathin slices were prepared with an ultramicrotome (Leica EM UC6) and collected onto Formvar-coated single-slot copper grids, then counterstained with lead citrate (Ultrostain, Leica, Wetzlar, Germany). The samples were investigated using a transmission electron microscope (Hitachi H-7100) and the perisomatic synapses labeled by the chromogens were identified.
3
Mouse Optic Nerve Ultrastructural Analysis
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Mouse optic nerves were prepared after deep anesthesia (isoflurane) perfusion fixation and immersion fixed (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.4.) for 24 h at 4°C. After tissue trimming and washes in 0.1 M sodium cacodylate buffer, postfixation in reduced osmium (2% osmium, 2.5% potassium ferrocyanide in 0.1 M cacodylate buffer) was followed by en bloc uranyl acetate (1% aqueous uranylacetate) contrasting, graded dehydration in ethanol and embedding in epon resin (Serva). After ultrathin sectioning the grids (Leica UC7 ultramicrotome) were contrasted by 1% uranyl acetate and Ultrostain (Leica). Images were acquired with a JEOL JEM1400 plus TEM equipped with a Ruby 8 Mpx CCD camera.
4
Ultrastructural Analysis of Microglia
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The microglial pellet was preserved throughout all fixation, contrasting and infiltration steps. Cells were fixed for 30 min in 2% PFA/2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M sodium cacodylate buffer (pH 7.4) (Sigma Aldrich), washed three times in 0.1 M sodium cacodylate buffer before postfixation in reduced osmium (1% osmium tetroxide (Science Services)/0.8% potassium ferrocyanide (Sigma Aldrich) in 0.1 M sodium cacodylate buffer). After contrasting in aqueous 0.5% uranylacetate (Science Services), the pellet was dehydrated in an ascending ethanol series, infiltrated in epon (Serva) and cured for 48 h at 60 °C. Ultrathin sections (50 nm) were deposited onto formvar-coated copper grids (Plano) and postcontrasted using 1% uranyl acetate in water and ultrostain (Leica). Transmission Electron Microscopy micrographs were acquired on a JEM 1400plus (JEOL) using the TEMCenter and tile scans with the ShotMeister software packages (JEOL), respectively. Three independent cell preparations were analyzed (n = 3).
5
Ultrastructural Analysis of Mouse Brain Tissue
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Mice brains were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 after deep anesthesia (isoflurane) perfusion. Brains were vibratome sectioned and immersion fixed in the same buffer for 24 h at 4 C. After tissue trimming and washes in 0.1 M sodium cacodylate buffer, postfixation in reduced Osmium (2% Osmium, 2.5% potassium ferrocyanide in 0.1 M cacodylate buffer) was followed by en bloc uranyl acetate (1% aqueous uranylacetate) contrasting, graded dehydration in ethanol and embedding in epon resin (Serva). After ultrathin sectioning the grids (Leica UC7 ultramicrotome) were contrasted by 1% uranyl acetate and lead citrate (Ultrostain, Leica). Images were acquired with a JEOL JEM1400 plus TEM equipped with a Ruby 8Mpx CCD camera. For each analysis, randomly selected regions in three to five different animals were imaged. Data analysis was carried out using ImageJ 1.41.
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