Technical steps towards enhanced localization of proteins in cultural heritage samples by immunofluorescence microscopy and micro-reflectance imaging spectroscopy

This study presents the results obtained during the optimization phase of a protocol based on immunofluorescence microscopy (IFM) with the aim to characterize and localize proteins in fluid-rich samples of cultural heritage interest. Chicken serum albumin from chicken blood was considered the primary target. Ovalbumin from chicken egg albumen (egg white) was also used as a common target protein to test the versatility of the protocol. Mock-ups were created, obtaining a complex distribution of the proteins in a porous matrix, thus providing a good approximation to a real case scenario. Thin sections of 1 µm thickness were then obtained using a cyanoacrylate-based tissue adhesive for the first time in samples of cultural heritage interest. Thin sections were preferred over standard cross sections, as they enabled multiple experiments to be performed by easily generating multiple sections from the same sample. This was particularly important during the optimization phase of the indirect immunochemical procedure, as the concentration of the blocking solutions, primary and secondary antibodies had to be optimized as well as the washing steps, time and temperature of incubation. The use of secondary antibodies conjugated with quantum dots fluorophores and the use of single wavelength structured illumination in the fluorescence microscope set-up reduced the contribution from unspecific fluorescence. All these technical advances led to the enhanced mapping of the distribution of both ovalbumin and chicken serum albumin in the mock-up samples. The material distribution was also mapped using micro-reflectance imaging spectroscopy, which confirmed the localization of the target materials, thus laying the foundation for a multi-modal imaging protocol, which will be applied to samples taken from fluid-rich patinas on West African sculptures.