A convenient method for in situ processing of cultured cells for cytochemical localization by electron microscopy

Abstract

The effects of various exogenous agents on subcellular structures is of importance to many investigators and can be critically evaluated by the use of cytochemical techniques and transmission electron microscopy. Therefore various cell culture techniques have become increasingly important in biological research in order to help determine these effects. Presently, most existing methods for processing anchorage-dependent cultures for electron microscopy utilize cells grown on either glass or plastic substrates. Therefore various coating substances have been applied to the culture surfaces to facilitate removal of the sample, however, incomplete separation and sample fracture often results. Here we present a simple method of in situ processing of samples for electron microscopy involving the use of detachable chamber slides. This method allows for the use of a quick processing procedure that results in a complete separation of the sample from the glass slide.

Mineralized nodule formation by human dental papilla cells in culture

“…By day 35, samples from the 3 groups were fixed in 3% glutaraldehyde and 0,01 M PBS (pH 7.4) at 4°C for 2 h, postfxed with 1% osmium tetroxide in the same buffer at 4°C for 1 h, dehydrated in a graded series of acetone, and embedded in Epon 812, The cells were removed from the glass slide according to a convenient method for in situ processing of cultured cells (8). Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate before examination in a Hitachi JEM-2000EX electron microscope.…”

In vitro διεύρυνση της εκκριτικής δραστηριότητας των αδενωμάτων υπόφυσης

“…Εκτός από την άμεση μικροσκόπηση, τα κύτταρα μπορούν να συλλεχθούν, να επιστρωθούν, να μονιμοποιηθούν και να χρωσθούν με ιστοχημικές αλλά και ανοσοϊστοχημικές μεθόδους. Επιπλέον είναι δυνατή η ανάλυση της μορφολογίας των καλλιεργούμενων κυττάρων με το ΗΜ ακολουθώντας ειδική επεξεργασία, είτε αφού προηγηθεί συγκομιδή τους από την καλλιέργεια (Thodou et al, 1995b), είτε με την ανάπτυξη τους α πό αρχής πάνω σε ειδικά καλλιεργητικά υποστρώματα που διευκολύνουν την in situ μονιμοποίηση και εμπέδωση τους (Kawamoto et al, 1980Kuhn, 1981, Ballou and Simpson, 1985.…”