H 7100 electron microscope

Transmission electron microscopy (TEM) was performed as previously described [3 (link)] by using 1% phosphotungstic acid (PTA)-stained bacteria on a carbon-coated grid and TEM pictures were obtained with a Hitachi H-7100 electron microscope (Hitachi High-Tech America, Pleasanton, CA, USA).

Renal cortex (1 mm³) from each rat was cut into small pieces and fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at 4°C for several days. After washing in phosphate buffer and postfixing in 1% OsO₄ for 2 h, the fixed material was dehydrated and embedded in Epon 812 (Okenshoji, Tokyo, Japan). Ultrathin sections were prepared and stained with uranyl acetate and lead citrate and examined with a Hitachi H7100 electron microscope (Hitachi, Yokohama, Japan).

TEM was performed as described by Caiola et al.[20] with some modifications. Overnight bacterial cultures were diluted 1∶100 in LB broth and incubated at 37°C for 4 h. Then, a total of 500 µl bacterial culture was washed and resuspended in 100 µl of PBS, from which 10 µl of the bacterial suspension was applied onto a carbon-coated grid (CF300-Cu, Electron Microscopy Sciences). After 20 min, excess solution was removed by a filter paper. Let the grid dry for additional 10 min. Finally, 1% phosphotungstic acid (PTA, 10 µl) was applied to the grid, staining for 15 sec and excess PTA was removed. On the next day, TEM pictures were obtained with a Hitachi H-7100 electron microscope.

P60 Het and WT kidney tissues stored in 3% glutaraldehyde were processed and embedded by the Department of Pathology, Tulane University. Ultimately, 60 nm sections were cut and imaged using a Hitachi H‐7100 electron microscope. Glomerular number was counted in Het and WT kidneys on P60 from 3 consecutive H&E‐stained sections/kidney adjacent to the longitudinal midplane (n = 3 kidneys/genotype). E13.5‐E15.5 4‐μm kidney sections from Mut and WT mice were processed for immunofluorescence using anti‐amphiphysin (ProteinTech, 1:200), anti‐active β‐catenin (ABC, Millipore, 1:400, anti‐Lotus Tetragonolobus Lectin (LTL) (1:400, Vector Laboratories), and anti‐cytokeratin (1:200, Sigma) antibodies. Immunostaining was performed by the immunoperoxidase technique with Vectastain Elite kit (Vector Laboratories, Burlingame, CA). Secondary antibodies were detected with Alexa Fluor dyes (Invitrogen). Specificity of immunostaining was documented by the omission of the primary antibody.

To prepare the cells for transmission electron microscopy (TEM), HT-29 cells were seeded at a density of 10⁶cells/T-25 ml flask and incubated overnight. The cells were then treated with the IC₅₀ of clausenidin (13.8 μg/mL) in a time dependent manner while the negative control cells were treated with 0.1 % (v/v) DMSO. After treatment, cells were harvested and washed with PBS before successive fixing with 4 % glutaraldehyde for 24 h and 1 % osmium tetraoxide at 4 °C for 2 h. After each fixing, washing was done three times with 0.1 M sodium cacodylate buffer. Dehydration of the cells was carried out with increasing concentrations of acetone (30 – 99.9 %). Further processing of cut sections was done using the method described by Tan et al. [23 (link)]. The sections were stained with uranyl acetate and viewed under the Hitachi H-7100 electron microscope.

Informed consents for collection of samples during surgery and autopsy and their subsequent use for genetic analysis and other research purposes were obtained from the patient’s family.
The surgical and autopsy specimens were fixed with 20% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-μm-thick sections stained with hematoxylin and eosin (HE), and the Klüver-Barrera method. The pathological diagnosis was made on the basis of the WHO classification of tumors of the central nervous system (CNS) by an experienced pathologist (AK). Immunohistochemistry (IHC) was performed as described previously using primary antibodies against Ki-67 (1:100, monoclonal, clone MIB-1, DAKO, Glostrup, Denmark), BRAF V600E (1:50, monoclonal, clone VE1, Spring Bioscience, Pleasanton, CA, USA) [18 (link), 19 (link)] and phosphorylated ERK (pERK: 1:200, monoclonal, 9101, Cell Signaling Technology (CST), Danvers, MA, USA).
Surgically obtained brain tissue and orthotopic brain tumor tissue were also subjected to electron microscopy. Glutaraldehyde-fixed small tissue blocks were post-fixed with 1% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in Epon 812. Ultrathin sections were then cut and stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7100 electron microscope at 75 kV.