Polysine slide

After electrophysiological recordings, midbrain samples were postfixed overnight in a 4% paraformaldehyde solution and then cryoprotected through immersion in a 30% sucrose solution. Using a cryostat apparatus (Leica CM1850UV), brains were cut into coronal sections (30 μm of thickness) and stored sequentially in 0,1 M PB containing 0.02% sodium azide. We performed a DAB (3,3′-diaminobenzidine) immunohistological staining using the Ultratek HRP Anti-Polyvalent Staining System (ScyTek Laboratories), according to the manufacturer’s instructions. Briefly, sections were incubated with mouse anti-tyrosine hydroxylase (TH) primary antibody in PB-Triton X-100 0.3% for three overnight at 4°C (1:1,000; Merck Millipore MAB318). Finally, the sections were mounted on polysine slides (Thermo Fisher Scientific, Waltham, MA, USA) and then dehydrated and coverslipped with Eukitt (Sigma).

Skin biopsies were fixed in 10% formalin for 24 h. Paraffin-embedded 5-μm-thick sections were cut, mounted on POLYSINE Slides (Thermo Fisher Scientific), dewaxed in xylene, and then dehydrated using an ethanol series. Hematoxylin and eosin (H&E) staining was performed to examine histological features and skin thickness. Toluidine blue (TB) staining was performed to visualize skin mast cells. Additionally, tissue slides were stained with primary antibodies listed in Table 1. The slides were washed with phosphate buffered saline with Tween 20 (PBS-T) and incubated with a two-component high-sensitivity 3,3-diaminobenzidine (DAB) chromogenic substrate (Vector Laboratories USA). After washing, the slides were dehydrated and mounted using a permount mounting medium (Thermo Fisher Scientific). All stained tissue slides were photographed using a slide scanner (Pannoramic MIDI; 3DHISTECH Ltd, Hungary) and analyzed using the Case Viewer software.

Shoot tips were collected from tobacco plants at weekly intervals, fixed overnight at 4°C with 4% paraformaldehyde in PBS (137 mM NaCl, 2.7 mM, 10 mM Na₂HPO4, 2 mM KH₂PO4, pH 7.4) and dehydrated in an incremental (30, 50, 70, 85 and 99%) ethanol series. Samples were cleared in Histo-clear (Natural Diagnostics, Atlanta, GA, USA) and embedded in paraffin (Paraplast; Sigma-Aldrich, St. Louis, MO, USA). Semithin longitudinal sections (10 µm) were collected on polysine slides (Thermo scientific, Braunschweig, Germany) and incubated for 16–18 hours at 37°C. For immunolocalization, sections were treated with Histo-clear for 3 min to remove paraffin, hydrated in ethanol series, pre-incubated with PBS containing 4% bovine serum albumin (BSA) for 30 min and incubated for 3 h at room temperature with a polyclonal serum raised in rabbit against AILV-V diluted 1∶500 in PBS. After washing in PBS three times, the samples were incubated with the secondary mouse-anti rabbit monoclonal antibody conjugated with alkaline phosphatase (Sigma Aldrich, St. Louis, MO, USA) diluted 1∶500 in PBS/BSA buffer and stained in NBT/BCIP solution (Sigma-Aldrich) following the protocol of the manufacturer. Slides were observed with a light microscope and pictures taken with a camera integrated to the microscope.

Mice were decapitated and skin and bones removed. The head was split with a razor blade 1 mm beside the sagittal midline to expose one nasal cavity. Turbinates, nasal septum or bulbs were carefully removed, and fixed for 10–30 min at room temperature. The tissue was collected in Ringer’s solution without touching the epithelial surface. Epithelium sheets were carefully transferred to an adhesion slide (Polysine® slides, Thermo Scientific) by moving it with fine forceps and fixed for 10 min at room temperature. Top view preparations were handled very cautiously to prevent damage of fine cilia structures. All solutions were applied by pipetting next to the tissue (Oberland and Neuhaus, 2014 (link)). Antibody staining was performed for 3 h or o.n.

Testicular tissues from 6 dpp mice and cultured explants were fixed in Bouin’s solution for 2 h at room temperature (RT). They were then dehydrated in graded series of ethanol and xylene in the Citadel 2000 tissue processor (Shandon, Cheshire, UK) and embedded in paraffin. Tissue Sections (3 µm thick) were cut using a microtome (JungRM 2035; Leica Microsystems) and mounted on Polysine slides (Thermo Fischer Scientific, Saint-Aubin, France). Tissue sections were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol.
Hemalun eosin saffron (HES) staining was performed to visualize tissue structural integrity and assign a global lesional score on a scale from 0 to 10 (0–5 for epithelial integrity and 0–5 for nuclear alteration, with 0 representing the complete absence of alteration and 5 representing the most important damage)^{49 (link)}.