I80 microscope

UV/Vis absorption spectra were recorded on a Hewlett-Packard mod. 8453 diode array spectrophotometer. CD spectra were obtained on a JASCO J-720 spectropolarimeter, equipped with a 150 W xenon lamp. To quantify the sign and magnitude of the supramolecular chirality we used the dissymmetry factor Δg = g(489 nm) − g(483 nm), where g is the ratio of the CD to the conventional absorption Optical micrographs were recorded with a Nikon i-80 microscope equipped with cross polarizers. AFM imaging was performed on a Multimode 8 microscope r(Bruker, USA). Samples were scanned at 1.1 Hz/line in PeakForce mode using Scanasyst-Air probes (Bruker, USA) in air, using an applied force of 2.5 nN. Image levelling and surface analysis were performed by Gwyddion 2.37 (http://gwyddion.net/). Stripes thicknesses were measured from topographic heights distribution.

FISH was performed according to the method described previously with some modification [73 (link)]. Paraffin sections were dewaxed and rehydrated in an ethanol gradient to water. The tissue sections were incubated with 5 μg/mL TexasRed-conjugated EUB338 (5′- GCTGCCTCCCGTAGGAGT-3′, Invitrogen) in hybridization buffer (0.1M Tris-HCl, 0.9 M NaCl, 0.1% SDS and 10% formamide, pH 7.2) at 40 °C overnight. The sections were rinsed in washing buffer (20 mM Tris-HCl, 0.9 M NaCl, pH 7.4) at 40 °C for 15 min and stained with 1 μg/mL DAPI. After staining, the sections were mounted with ProLong Gold mounting medium (Invitrogen). All images were obtained and analyzed with a Nikon i80 microscope.

Paraffin embedded sections were dewaxed with xylene and rehydrated in graded series of ethanol. Tissue sections were then washed with PBS before starting staining procedure. Immunohistochemistry was done using Histostain-SP kit (Invitrogen, Carlsbad, CA, USA) and following their instructions. Briefly, deparaffinized sections were boiled for 20 minutes in 0.01 M citrate buffer at pH 6.0 for epitope retrieval. The sections were then blocked for 10 minutes using a serum blocking solution. Sections were incubated for 1 hour at 4°C with primary antibodies (described below). After 3 washings of 5 minute each in PBS, sections were subsequently incubated with biotinylated secondary antibodies for 10 minutes, followed by 10-minute incubation with enzyme streptavidin-peroxidase conjugate. This was followed by 3-5 minute incubation with DAB solution containing diaminobenzidine (DAB) chromogen for developing and 0.6% H₂O₂ to inhibit endogenous peroxidase activity. The sections were finally counterstained with hematoxylin and dehydrated with graded series of ethanol and cleared in xylene. After staining, the sections were mounted with mounting medium and images were obtained and analyzed with a Nikon i80 microscope.