Induced AECs on the PET membrane before transplantation and F344 wild-type rat middle ear mucosa were observed using scanning and transmission electron microscopes. For scanning electron microscopy (S-4700 [Hitachi Co., Tokyo, Japan]), the cells were fixed with 4% paraformaldehyde (PFA) (Nacalai Tesque)/2% glutaraldehyde (FUJIFILM Wako Pure Chemical Corporation) in phosphate buffer at 4 °C overnight. Then, the cells were post-fixed in 1% osmium tetroxide (Nacalai Tesque), dehydrated, dried using a critical point drying method, and coated with platinum palladium. For transmission electron microscopy (H-7650, Hitachi Co.), the fixed cells were post-fixed in 1% osmium tetroxide for 2 h and dehydrated using an ethanol series. The cells were then embedded in epoxy resin and DMP-30 (Nacalai Tesque). Thin sections were stained with uranyl acetate (Merck Millipore, Burlington, MA, USA) and lead citrate (Nacalai Tesque).
Lead citrate
Manufactured by Nacalai Tesque
Sourced in United States
Lead citrate is a chemical compound used as a staining agent in electron microscopy. It is a white to pale yellow crystalline powder that is soluble in water. Lead citrate is primarily used to enhance the contrast of biological samples in transmission electron microscopy (TEM) imaging.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Fujifilm (2 mentions)
S 4700, by Hitachi (1 mentions)
Uranyl acetate, by Merck Group (1 mentions)
H 7650, by Hitachi (1 mentions)
Osmium tetroxide, by Nacalai Tesque (1 mentions)
Em uc7, by Leica (1 mentions)
Phosphate buffer, by Fujifilm (1 mentions)
Propylene oxide, by Fujifilm (1 mentions)
Uranyl acetate, by Fujifilm (1 mentions)
Jem 1400plus electronic microscope, by JEOL (1 mentions)
Epon 812, by TAAB (1 mentions)
2 protocols using lead citrate
1
Ultrastructural Analysis of AECs on PET Membrane
2
Ultrastructural Analysis of Cellular Samples
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Cellular samples were fixed in 0.9% NaCl, 1% glutaraldehyde (Wako) for 1 h at 4 °C. Cells were pelleted and re-suspended in 1 mL of a 37 °C pre-warmed 1.1% agarose solution (Bio-Rad). After centrifugation (3000 rpm at 37 °C), pellets were placed at 4 °C and cut into small specimens, washed in 5% sucrose PBS (Wako) and post-fixed with 5% sucrose, 1.5% osmic acid (Nisshin EM) in 100 mM phosphate buffer (Wako) pH 7.3, for 1 h at 4 °C. Post-fixed specimens were washed in 5% sucrose PBS and dehydrated in a graded series of ethanol (Wako) and propylene oxide (Wako), then embedded in epoxy resin EPON 812 (TAAB Laboratories). Ultrathin sections were obtained with an ultra-microtome (Leica EM UC7) and subjected to double staining with a uranyl-acetate (Wako) and lead-citrate (Nacalai Tesque). Samples were observed under a JEM-1400Plus Electronic Microscope (JEOL). TEM were analyzed by collecting images of at least 3 representative areas (RA) at magnification of ×500. One RA contained 10–20 nucleated cells. Then representative images were taken at higher magnifications.
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