Indirect Immunofluorescence

The number of organisms or latex beads that were endocytosed or cell-associated with the various host cells was determined using our standard differential fluorescence assay [5 (link),7 (link),9 (link),51 (link)]. The host cells were grown to 95% confluency onto 12-mm diameter coverslips coated with fibronectin in 24-well tissue-culture plates. They were incubated with 10⁵C. albicans hyphae in RPMI 1640 medium. After 45 min, the cells were rinsed twice with Hank's balanced salt solution (HBSS; Irvine Scientific) in a standardized manner and then fixed with 3% paraformaldehyde. In experiments performed with C. albicans, the adherent but nonendocytosed organisms were labeled with rabbit polyclonal anti–C. albicans antibodies (Biodesign International, http://www.biodesign.com) that had been conjugated with Alexa 568 (Invitrogen), which fluoresces red. Next, the cells were permeablized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS, and then the cell-associated organisms (the endocytosed plus nonendocytosed organisms) were labeled with the anti–C. albicans antibodies conjugated with Alexa 488 (Invitrogen), which fluoresces green. The coverslips were viewed using an epifluorescent microscope, and the number of endocytosed organisms was determined by subtracting the number of nonendocytosed organisms (which fluoresced red) from the number of cell-associated organisms (which fluoresced green). At least 100 organisms were examined on each coverslip, and the results were expressed as the number of endocytosed or cell-associated organisms per high-powered field.
Experiments investigating the endocytosis and adherence of the yellow-green fluorescing latex beads were performed similarly, except that 3 × 10⁵ beads were added to each well. The adherent, nonendocytosed control beads coated with biotinylated BSA were labeled with strepavidin Alexa 568. The adherent beads coated with either rAls1-N or rAls3-N were detected by indirect immunofluorescence using rabbit polyclonal anti-Als1 antibodies followed by Alexa 568–conjugated goat anti-rabbit antibodies.

Deep-Manager platform allows users to perform specific sensitivity tests to their own images dataset to select the most appropriate features for the specific classification task. Sensitivity tests aim to detect which features extracted from ad hoc algorithms (handcrafted) or from a pre-specified Deep Learning network through transfer learning approach are more sensitive to external quantities and phenomena that are acquisition-specific. Among the existing vast panorama of acquisition devices and experimental set-up, with the aim to prove the effectiveness of the proposed method, we selected three of the most used practical contexts in the field of biological image analysis: 2D transmission light time-lapse microscopy, 3D phase-contrast time-lapse microscopy, and 3D fluorescence time-lapse microscopy. The implemented sensitivity tests are therefore thought for those contexts. However, the list of possible tests of the Deep-Manager platform could be enlarged in the future to other fields such as histopathological imaging or indirect immunofluorescence. For this reason, in the remainder, we will indicate the present release as Deep-Manager 1.0 version. Link: https://github.com/BEEuniroma2/Deep-Manager. All the data required to reproduce the figures are in the Supplementary Data 1 file.

Indirect immunofluorescence was used to identify OPCs (NG2 or PDGFαR-positive cells) and mature (CNPase-positive cells) and myelinating (MBP-positive cells) oligodendrocytes. Cultures were fixed in 4% of cold paraformaldehyde for 15 min and all primary and secondary antibodies used in the present study are listed in Table 1. For AKT/pAKT identification, during fixation a preotease/phosphatase inhibitor cocktail (PMSF 1 mM, sodium floride 10 mM, sodium orthovanadate 1 mM) was added. Cells were also incubated with the nuclear dye Hoechst 33258 (1 μg/mL in PBS, 0.3% Triton-X 100) to identify the nuclei.
For each reaction a control group stained only with secondary antibodies was included to check for specificity.