Cell surface protein detection with immunogold labelling in ESEM: Optimisation of the method and semi‐quantitative analysis

Abstract

In this work we used a combination of immunogold labelling (IGL) and environmental scanning electron microscopy (ESEM) to detect the presence of a protein on the cell surface. To achieve this purpose we chose as experimental system 3T3 Swiss Albino Mouse Fibroblasts and galectin-3. This protein, whose sub-cellular distribution is still under discussion, is involved in a large number of cell physiological and pathological processes. IGL technique has been utilised by many authors in combination with SEM and TEM to obtain the identification/localisation of receptors and antigens, both in cells and tissues. ESEM represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artefacts and interfere with IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labelling detection is based on the atomic number difference between Nanogold spheres and the biological material. Using the gaseous secondary electron detector (GSED) compositional contrast is easily revealed by the backscattered electrons component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimised to improve the intensity and the specificity of the labelling signal, in order to obtain a semi-quantitative evaluation of the labelling signal.

Randomized comparison of reduced fat and reduced carbohydrate hypocaloric diets on intrahepatic fat in overweight and obese human subjects

“…In a previous work (Muscariello et al, 2008), we showed that the high resolution power of electron microscopy in ESEM modality is able to evaluate Gal‐3 expression on the cell surface also on Thin‐prep treated human biological samples.…”

“…The first class (0–2 spots/cell) was considered negative (unlabeled sample), since all methods, including ESEM‐IGL, have a percentage of non‐specific labeling (Muscariello et al, 2008). The remaining four classes were obtained assuming a linear progression of about 7 spots/cell.…”

“…In our search to optimize the method and demonstrate the specificity of the marker, we used 3T3 cells (Swiss Albino mouse fibroblasts) as a Gal‐3 positive control and D16 cells (murine mesoangioblasts) as a negative control. In this way, the marking procedure has been optimized and the risk of false positives, false negatives, and background has been minimized (Muscariello et al, 2008).…”

“…In our study, we used the technique of Immunogold Labelling (IGL) to detect membrane Gal‐3 expression (Cafiero et al, 2011; Muscariello et al, 2008). Briefly, IGL is a method similar to classic immunocytochemistry, but the secondary antibody is conjugated to gold nanoparticles (in our case 1.4 nm in diameter, to minimize a specific binding).…”

Nano needle with buffering beam for single cell stiffness measurement by nanorobotic manipulators inside ESEM

“…Moreover, ESEM has a wider working space for manipulation comparing with AFM, and the observation is not restricted to the contact with sample. Therefore, ESEM offers new opportunities for single cell analysis at nanoscales [6], [7]. This system has been used to observe and characterize the property of bio samples [8].…”