Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates.

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.

Microscopy of the Drosophila facet eye: vademecum for standardized fixation, embedding, and sectioning.

We describe here a standardized method for histological processing of the Drosophila compound eye. Primary fixation with 2.5% glutaraldehyde, obligatorily supplemented with 0.1% household detergent regularly yielded the best structural preservation, as compared with that of other, more complicated fixation protocols tested. Notably, it proved indispensable not only to cut off the fly's head to facilitate the penetration of the reagents but also to open the chitinous head capsule. For this, we locally pierced the cuticle between the eyes, leaving the head structurally almost intact, a prerequisite for precisely aligning the head for microtomy. We developed a two-step re-embedding procedure allowing for exact and reproducible orientation of the fly heads. Thus, highly comparable series of cross sections through a representative number of ommatidia were obtained. The feasibility of our embedding and sectioning approach is finally demonstrated by three-dimensional reconstructions of the middle segments of the R1, R7, and R8 photoreceptor cells. We present reconstructions from structurally modified ommatidia, as seen after RNAi-mediated depletion of the endosomal adaptor protein p14, and from normal ommatidia corresponding to the wildtype.