Preparation of cultured mammalian cells for transmission and scanning electron microscopy using aclar film

Abstract

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated-chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical-point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high-resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.

Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

“…For immuno-EM, cell lines were grown for 3-4 days on Aclar film prior to embedding in Lowicryl K4M as described (14,15). Thin sections were labeled with 0.1 mg of mAb 1D4 per ml for detection of wt rhodopsin and the P23H § mutant or with 0.025 mg ofa polyclonal sheep anti-opsin antibody per ml, which primarily detects the amino termini of vertebrate rhodopsins (16) for detection of wt rhodopsin and mutants P347L and Q344ter (Gln-344 --termination-i.e., deletion of C-terminal positions 344-348).…”

“…For fluorescent staining, cells were fixed for 10 min at room temperature in 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized for 3 min with ice-cold methanol, and incubated with mAb B6-30 or 1D4 (ascites diluted 1:5000), followed by fluorescein isothiocyanate-conjugated goat anti-mouse Ig. For avidin-biotin complex (ABC)-peroxidase staining, cells were rinsed with PBS, air-dried, fixed and permeabilized for 10 min with ice-cold methanol, and incubated with mAb B6-30 or 1D4 followed by biotinylated goat anti-mouse immunoglobulin.For immuno-EM, cell lines were grown for 3-4 days on Aclar film prior to embedding in Lowicryl K4M as described (14,15). Thin sections were labeled with 0.1 mg of mAb 1D4 per ml for detection of wt rhodopsin and the P23H § mutant or with 0.025 mg ofa polyclonal sheep anti-opsin antibody per ml, which primarily detects the amino termini of vertebrate rhodopsins (16) for detection of wt rhodopsin and mutants P347L and Q344ter (Gln-344 --termination-i.e., deletion of C-terminal positions [344][345][346][347][348].…”

Endocytosis of Ultrafine Particles by A549 Cells

“…Aclar embedding sheets (Ted Pella, Inc., Redding, CA) were cut into circles ( Ͻ 16 mm diameter) and notched on one edge to indicate orientation (27). They were sterilized in 70% alcohol for 0.5 h and air-dried before placement in sterile 24-well plates and the addition of A549 cells.…”

Patterned poly(chlorotrifluoroethylene) guides primary nerve cell adhesion and neurite outgrowth

“…Unlike fluoropolymer surfaces, such as FEP, 31 PCTFE surfaces supported minimal nerve cell adhesion, which also has been observed by others. 16 Following PEG modification, the number of adherent cells decreased significantly (p < 0.05), confirming the utility of this surface for patterning. There was a dramatic increase in cell adhesion following peptide modification of gold.…”