Pfa pb

Morphological changes in the cell nucleus were examined by DAPI staining. Cells treated with 3OTPCA were washed with PBS and fixed with 4% paraformaldehyde (PFA/PBS (v/v); Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 4°C. Then, cells were stained with 2 μg/mL DAPI (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 5 min for nuclear visualization and thoroughly washed before observation under a fluorescent microscope (GE Healthcare Delta Vision Elite) [16 (link)].

Seventy-five thousand COS7 cells were seeded on glass coverslips in 24-well plates and were transfected 16 h growth later with 250 ng of pEXP-VC155(Nter) and 250 ng of pEXP-VN173(Nter) plasmids encoding HOXA1 and ERα fusion proteins, respectively. Empty pDEST-VC155(Nter) and pDEST-VN173(Nter) vectors were used as negative controls. Twenty-four hours post-transfection, cells were washed twice with PBS and fixed for 20 min with 4% PFA-PBS (#441244, Sigma-Aldrich) at room temperature. Cells were then rinsed twice for 5 min in TBS-T buffer (50 mM Tris-HCl, pH 7.5, 155 mM NaCl, 0.1% Triton X-100 (#10789704001, Merck)) and once for 10 min with TB buffer (50 mM Tris-HCl, pH 7.5). Cells on coverslips were stained in a mounting medium containing DAPI and Vectashield (#H-1200, Labconsult, Brussels, Belgium), and pictures were taken under an epifluorescence microscope (Axioskop 2, Zeiss, Oberkochen, Germany). Fluorescence was quantified with the IMAGEJ software and tested interactions were considered as positive when the emitted fluorescence was at least three times higher than in the negative control conditions. pEXP-VN173(Nter)-hHOXA1 with pEXP-VC155(Nter)-mHOXA1 was used as a positive BiFC control in each experiment.

Tissues were embedded in Tissue‐Tek OCT medium and snap frozen in liquid nitrogen. For immunofluorescence, 10‐μm cryosections were rehydrated to water, postfixed with 4% PFA/PBS (Sigma‐Aldrich), blocked and permeabilized for 30 min in .25% Triton X‐100/5% normal donkey serum (NDS)/PBS (Vector Laboratories). Antibodies were diluted in 5% NDS/.1% Tween‐20/PBS. The following primary antibodies were incubated at 4°C overnight: anti‐FAPα (AF3715, R&D Systems), anti‐CXCL12 (MA5‐23759, Thermo Fisher Scientific), anti‐THBD (hpa002982, Atlas Antibodies), anti‐NPM1 (FC61991, Thermo Fisher Scientific), anti‐FBLN1(hpa001612, Atlas Antibodies), anti‐CD90 (AF2067, R&D Systems), anti‐c‐Myc (MAB36961, R&D Systems) and anti‐c‐Fos (PC05L, Merck). Staining was visualized with secondary antibodies (IgG‐NL493 (NL006) and IgG‐NL557 (NL010), 1:200, R&D Systems). Nuclei were visualized using ProLong Gold Antifade Mountant (P36931, Thermo Fisher Scientific). Stains were evaluated with a Leica DM6000 fluorescence microscope and using LAS AF Lite software.

Hooves were obtained from horses that were not euthanized for research purposes. Horse hooves were collected from the abattoir 1 h post euthanasia following ethical approval by The School of Veterinary Medicine and Science, University of Nottingham. For three-dimensional imaging and histological sampling, PBS was injected through the medial and lateral palmar digital arteries to remove the blood, and tissues were fixed by replacing PBS with a 4% PFA/PBS (Sigma, UK) fixative solution under manual pressure. When needed, biopsies were taken from the dorsal and quarter parts of the coronary band and placed into a 4% PFA/PBS solution fixative solution prior processing. For primary cell isolation, hooves were aseptically cleaned and progenitor keratinocyte cells obtained as described below. All primary cell cultures were performed at 37°C in 5% CO₂.

HGMVECs and BOECs were seeded in a black 96-well plate with a clear bottom (Falcon Corning Incorporated, Corning, NY, USA). Cells were incubated with either control medium or medium with 10 ng/mL of TNFα (Roche Diagnostics GmbH, Mannheim, Germany) for 24 h and fixed with 2% paraformaldehyde (PFA)/PBS (Sigma Aldrich) at room temperature for 15 min. Mouse anti-CD31 (BD Biosciences, San Jose, CA, USA) primary and corresponding species-specific Alexa Fluor 568 were used as antibodies. Alexa 488-labeled Stx-B at a concentration of 5 μg/mL and the fluorescent DNA stain DAPI (Invitrogen, Carlsbad, CA, USA) were added, and images were taken with the use of a Zeiss LSM900 confocal microscope (Zeiss, Oberkochen, Baden-Württemberg, Germany).