Uc7 rt ultramicrotome

After being treated with IL-37 for 48h, cells pellets were collected, and prefixed with 3% glutaraldehyde and refixed with 1% tetroxide. Then the pelleted were dehydrated with an acetone gradient (series of solutions from 30% to 100%), and permeated by a mixture of dehydrant and Epon812 embedding agent, following embedded with Epon812. Subsequently, the pelleted material was cut into thin (60-90nm) sections using a LEICA UC7rt ultramicrotome and collected onto copper mesh, then stained with uranyl acetate for 10-15 minutes and lead citrate for 1-2 minutes. The JEM-1400FLASH (LEOL, Japan) was used to observe the melanosomes.

The biofilm preparation for TEM analyses was performed as previously described (Melloul et al., 2016 (link)). Briefly, samples were fixed with 2.5% glutaraldehyde-cacodylate buffer (pH 6.5) and then post-fixed with osmium tetroxide and dehydrated with different dilutions of alcohol (50–100%). The samples were then embedded into EPON resin and left to polymerize. Ultra-fine sections were obtained using a Leica UC7-RT ultramicrotome, and contrasted with lead-citrate and uranyl-acetate solutions. Specimens were mounted on grids to be examined under the microscope (JEOL 100 CX II instrument, Japan). TEM was used to measure the cell wall thickness of A. fumigatus in single and mixed biofilms using ImageJ program. For both biofilms, between 10 and 20 measurements of cell wall thickness were performed on 15 different hyphae.

Biofilm samples for TEM were first incubated 10 min at 4°C and then fixed with a solution of 2.5% glutaraldehyde diluted in sodium cacodylate buffer (pH 6.5) for 3 min. The solution was removed and then a similar volume of 2.5% glutaraldehyde solution was added and the whole mix was incubated at 4°C for 20 min. The cultures were finally placed at 4°C in sodium cacodylate buffer (pH 6.5). The biofilm samples were then post-fixed with osmium tetroxide and dehydrated with different dilutions of alcohol (50–100%). The samples were then embedded into EPON resin and left to polymerize. Ultra-fine sections were obtained using a Leica UC7-RT ultramicrotome, and contrasted with lead-citrate and uranyl-acetate solutions. Finally, specimens were mounted on grids to be examined under the microscope (JEOL 100 CX II instrument, Japan). TEM was used to measure the cell wall thickness of A. fumigatus in single and mixed biofilms using the ImageJ program (http://imagej.nih.gov/ij/). For both biofilms, between 10 and 20 measurements of cell wall thickness were performed on 27 hyphae.