Elvanol

The expression of the SB3 protein in transfected clones was assessed by immunofluorescence. Cells were seeded on slides (5 × 10³ cells/well in a 12 well plate) and cultured overnight. Cells were then fixed in 4% paraformaldehyde, permeabilized with 0.2% Tryton X 100, and blocked with 5% Goat serum in 1% BSA in PBS. Slides were then incubated with 8 μg/mL of a rabbit polyclonal antibody against SB3 (Hepa-Ab, Xeptagen, Marghera, VE, Italy), washed with 0.1% Tween 20 in PBS, and incubated with 488 AlexaFluor anti-rabbit secondary antibody (1:500 dilution) (Invitrogen, Carlsbad, CA, USA).
Cellular nuclei were counter stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Slides were mounted with ELVANOL (Sigma-Aldrich, St. Louis, MO, USA) and observed under a fluorescence microscope utilizing the optical sectioning of Apotome.2 (Axiovert 200M, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) for high quality images.

HepG2 and HAT22/VGH were seeded on slides (4 × 10⁵ cells/slide) and cells were fixed with 4 % paraformaldehyde, permeabilized with 0.4 % Tryton X-100, and blocked with 5 % goat serum (Invitrogen Life Technologies, Waltham, MA, USA) in PBS containing 1 % BSA. Slides were incubated with rabbit monoclonal anti-C/EBP-β and anti-PAR2 (Abcam-Cambridge UK) antibodies for 1 h at room temperature, followed by incubation with the anti-rabbit Alexa Fluor 546 (Invitrogen Life Technologies, Waltham, MA, USA) secondary antibody. Cellular nuclei were counterstained with Dapi (Merck KGaA, Darmstadt, Germany). Slides were mounted with ELVANOL (Merck KGaA, Darmstadt, Germany) and observed under a fluorescence microscope (Axiovert 200M-Apotome.2, Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

Immunofluorescence studies were carried out on hepatoma cells (HepG2 cell line) (ATCC, Manassas, VA, USA) engineered to overexpress SerpinB3 (HepG2/SB3) by transfection with a plasmid expression vector containing the human SerpinB3 gene (pCDNA3/SB3), and on HepG2/Control cells containing the plasmid vector alone (pcDNA3.1D/V5-His-TOPOTM), obtained as previously described [31 (link)].
HepG2 cells were seeded on slides (4 × 10⁵ cells/slide) and cultured for 48 h. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.4% Tryton X-100 and blocked with 5% goat serum (Invitrogen, Waltham, MA, USA) in PBS containing 1% BSA. Slides were incubated with 2 μg/mL of anti-P#3antibody, anti-P#5antibody and with anti-SerpinB3/4 monoclonal antibody (OriGene Tech., Rockville, MD, USA) at 1:150 dilution for 1 h at room temperature, followed by incubation with the anti-rabbit Alexa-Goat 546 and anti-mouse Alexa-Goat 488 secondary antibodies (Invitrogen Life Technologies, Waltham, MA, USA). Cellular nuclei were counterstained with Dapi (Merck KGaA, Darmstadt, Germany). Slides were mounted with ELVANOL (Merck KGaA, Darmstadt, Germany) and observed under a fluorescence microscope (Axiovert 200M-Apotome.2, Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

HepG2/control cells were seeded on slides (4 × 10⁵ cells/slide) and cultured for 24 h. Cells were treated as follows: (a) overnight incubation with PBS as a negative control, (b) overnight incubation with 100 ng/mL of SerpinB3 recombinant protein as a positive control, (c) pre-treatment with 5 g/mL of the anti-LRP-1 85 kDa monoclonal antibody or with a generic mouse Ig for one hour followed by overnight incubation with 100 ng/mL of SerpinB3 recombinant protein, and (d) overnight incubation with 5 g/mL of RAP and 100 ng/mL of SerpinB3 recombinant protein. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.4% Tryton X-100, and blocked with 5% goat serum (Invitrogen Life Technologies, Waltham, MA, USA) in PBS containing 1% BSA. Slides were incubated with polyclonal anti-vimentin and anti-snail antibodies and with monoclonal anti-E-cadherin antibodies for 1 h at room temperature, followed by incubation with the Alexa-Goat 546 and 488 secondary antibodies, respectively. Cellular nuclei were counterstained with Dapi (Merck KGaA, Darmstadt, Germany). Slides were mounted with ELVANOL (Merck KGaA, Darmstadt, Germany) and observed under a fluorescence microscope (Axiovert 200M-Apotome.2, Carl Zeiss MicroImaging GmbH, Göttingen, Germany).