PR droplet (250μM PR20 + 0.04μg/μl polyU RNA) sample (3.5 μL) was applied to a 300 mesh lacey Quantifoil grid and incubated for 30 s. Next, excess buffer was removed by blotting the grids for 3 s using a Whatman 1 filter paper and the sample was snap frozen by plunging in liquid ethane at a temperature of −180°C and stored in liquid nitrogen until visualization. Next, the samples were transferred to a Gatan 914 cryoholder and imaged at low dose conditions at −177°C, using a JEOL JEM1400 TEM equipped with an 11 Mpxl Olympus SIS Quemesa camera.
Sis quemesa
Manufactured by Olympus
The SIS Quemesa is a laboratory equipment designed for spectroscopic analysis. It is capable of performing various types of spectroscopic measurements, including UV-Vis, fluorescence, and luminescence spectroscopy. The SIS Quemesa is a versatile instrument that can be used in a wide range of scientific applications, including chemistry, biology, and materials science.
Automatically generated - may contain errors
3 protocols using sis quemesa
1
Cryo-EM sample preparation protocol
2
Ultrastructural Analysis of HEK Cells and Neurons
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HEK cells and primary neurons grown on aclar coverslips were rinsed briefly and fixed in 1.3% glutaraldehyde in 66 mM cacodylate buffer for 2 h at RT before processing for electron microscopy. Then the cells were washed for 30 min in 0.1 M cacodylate buffer and post-fixed for 2 h at RT in 1% OsO4, 1.5% K4Fe(CN)6 in 0.1 M cacodylate buffer. Cells were rinsed again in 0.1 M cacodylate buffer and stained with 3% uranyl acetate for 1 h. Dehydration was performed at RT by transferring the cells to 35%, 50%, 70%, 90% and two steps of 100% ethanol for 10 min each. The cells were then embedded in EMbed812 and resin blocks were sectioned on an ultramicrotome (Leica Ultracut UCT, Leica Mycrosystems). Ultrathin sections (70 nm) were mounted on copper grids and imaged using a JEM-1400 transmission electron microscope (JEOL), equipped with an 11Mpixel Olympus SIS Quemesa camera. Quantifications of the area and number of subcellular compartments were performed with the image processing software ImageJ (Fiji distribution).
3
Multimodal Imaging Workflow for Xenograft Analysis
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Morphological Analysis for Quantification Morphometry and measurements were performed with Fiji/ImageJ software. For quantifications, at least 4-5 different brain sections comprising the human xenografts and the adjacent mouse host tissue were included per animal.
Immunofluorescence (IF) sections were imaged by confocal microscopy (Nikon Ti-E inverted microscope) with a 20x (0.75 NA) objective to image Z series stacks of areas inside human xenografts or mouse host tissue (8-10 optical sections 1 mm apart for each image). All images were acquired using identical acquisition parameters as 16-bit, 1024x1024 arrays. Z series stacks were then converted in Fiji/ImageJ to maximum intensity projections and threshold adjusted to isolate specific fluorescence.
Immunohistochemistry (IHC) sections were imaged using a Leica DM 2500M light microscope with a 63x objective. All images were acquired using identical acquisition parameters as 16-bit, 1024x1024 arrays.
Ultrathin EM sections comprising either the human xenografts or the adjacent mouse host tissue were imaged using a JEM-1400 transmission electron microscope (JEOL) equipped with a 11M-pixel Olympus SIS Quemesa camera. Images were taken in randomly selected areas at a 600x magnification.
Immunofluorescence (IF) sections were imaged by confocal microscopy (Nikon Ti-E inverted microscope) with a 20x (0.75 NA) objective to image Z series stacks of areas inside human xenografts or mouse host tissue (8-10 optical sections 1 mm apart for each image). All images were acquired using identical acquisition parameters as 16-bit, 1024x1024 arrays. Z series stacks were then converted in Fiji/ImageJ to maximum intensity projections and threshold adjusted to isolate specific fluorescence.
Immunohistochemistry (IHC) sections were imaged using a Leica DM 2500M light microscope with a 63x objective. All images were acquired using identical acquisition parameters as 16-bit, 1024x1024 arrays.
Ultrathin EM sections comprising either the human xenografts or the adjacent mouse host tissue were imaged using a JEM-1400 transmission electron microscope (JEOL) equipped with a 11M-pixel Olympus SIS Quemesa camera. Images were taken in randomly selected areas at a 600x magnification.
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