Tritc labeled secondary antibodies

Cultured neurons were washed and fixed in PFA (4% w/vol) for 20 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 in PBS for 15 min and incubated at room temperature for 1 h with rabbit polyclonal anti-peripherin antibody (Millipore, Milan, Italy). Following extensive washes in PBS, cells were incubated for 1 h at room temperature with TRITC labeled secondary antibodies (Molecular Probe, Milan, Italy). Nuclei were stained with TOTO-3 iodide (Molecular Probe). Samples were then analysed using Leica TCSNT/SP2 confocal microscope. All microscope parameters were set to collect images below saturation and were kept constant during acquisition.
Number of peripherin positive cells was recorded within a minimum of 3 randomly selected fields covering 1.254 mm² from five independent experiments. The percentage of peripherin positive cells was calculated on the number of total nuclei stained with TOTO-3 iodide. Axonal length was measured in stained neuronal cells by using ImageJ 1.40 g (National Institutes of Health; Bethesda, MD, USA). For each experimental condition, 10 cells in 8 images obtained from at least three separate staining sessions were considered. Quantitative analysis was performed manually by using a counting grid, as previously reported^{43 (link)}.

Fixation of cells was conducted as described above. Permeabilization was conducted with 0.25% Triton X-100/PBS for 7 min at RT. Double staining of target antigens was achieved by incubation with first primary antibody for 45 min. Followed by subsequent washing with PBS and incubation with the second primary antibody at the same conditions. Visualization of primary antibodies was conducted with Alexa488 or TRITC labeled secondary antibodies (Molecular Probes, Eugene, Oregon, USA) diluted at 1/200 with 1 μg/ml DAPI in antibody diluent (Zytomed Systems, Berlin, Germany) for 45 min. at room temperature. Unbound antibodies and DAPI were removed by washing with PBS before mounting of cover slips on microscopic slides (Super Frost Plus, Menzel Gläser, Germany) with DABCO. In general, each reagent step was followed by thorough washing with 200 μl of PBS. To detect cells of hAECII lineage, the surfactant protein transcription factor TTF-1 [19 (link)] was targeted with mouse anti-TTF-1 (clone SPT24, Zytomed Systems, Berlin, Germany) at 1/400 dilution. Activity of collagen production and secretion processes were assessed by targeting the collagen chaperon HSP47 [20 (link)] with rabbit anti-HSP47 (clone EPR4217, Abcam, Oxford, UK) at 1/100 dilution.