Estudios de patogenicidad

2.5.1 Immunohistochemistry

Archival human liver tissue was obtained with full ethical approval and written informed consent from the Newcastle & North Tyneside Research Ethics Committee.

Tissues were fixed in 10% (v/v) formalin (Fisher Scientific, Loughborough, UK) diluted in 1 x PBS for 24 hours and then processed through increasing concentrations of ethanol. Tissues were embedded in paraffin and sections were

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cut (4-8µm) and mounted onto super frost microscope slides and incubated overnight at 37o_C.

Tissue sections were de-waxed in xylene (Fisher Scientific, Loughborough, UK) for 10 minutes and then rehydrated in 100% and then 95% ethanol (Fisher Scientific, Loughborough, UK) for 3 minutes each and then placed into water. To break the protein cross-links formed by formalin fixation and to reveal antigenic sites an antigen retrieval process was carried out. Antigen retrieval was performed by immersing sections into 0.01M citrate buffer pH6 in a pressure cooker and timing for 1 minute once pressure of the system was reached. Tissue sections were then cooled with water, outlined with a hydrophobic pen and then incubated in 3% (v/v) hydrogen peroxide H2O2 prepared in methanol for 10

minutes to ensure endogenous peroxide activity was quenched. Tissue sections were then washed in 1 x PBS prior to blocking non-specific binding of antibodies with 20% FCS (v/v) in 1 x PBS for 30 minutes. Primary rabbit antibodies were diluted in 1 x PBS containing 0.05% (v/v) FCS (Table 2.7), added to each section and then incubated overnight at 4o_{C. Following overnight incubation, sections}

were washed 2 x for 5 minutes in 1 x PBS before being incubated for 1 hour at room temperature with the appropriate secondary HRP antibody (Table 2.7). Following a final 2 washes x 5 minutes in 1 x PBS, staining was developed using diaminobenzidine (DAB) (Dako UK Ltd, Cambridgeshire, UK) which is oxidized by hydrogen peroxide and a dark brown colour is observed. The DAB was added to each section (~200μl) and incubated for 1 minute and then washed in water prior to haematoxylin staining. Sections were counterstained with haematoxylin for 2 minutes, washed with water and then stained with Scotts tap water (Fisher Scientific, Loughborough, UK) for 30 seconds. Finally, sections were dehydrated briefly through a series of ethanols (50%, 75%, 95%, 100%) and then cleared in xylene for 10 minutes prior to mounting in Depex (DPX) (Sigma-Aldrich, Gillingham, UK).

2.5.2 Haematoxylin and Eosin (H & E) staining

Haematoxylin (stains the nuclei blue) and eosin (stains the cytoplasm red) is the most common staining method used in histology. Tissue sections were de-waxed and rehydrated as described in section 2.5.1. They were then stained in haematoxylin for 2 minutes, washed with normal tap water and then immersed in

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Scott’s tap water for 30 seconds. Tissue sections were then counter stained in eosin (Fisher Scientific, Loughborough, UK) for 30 seconds, washed in water and then dehydrated and mounted as described in section 2.5.1.

2.5.3 Sirius red staining

Picro-sirius red binds to collagen due to its sulphonic groups reacting with the basic groups found in collagens and so is ideal for determining levels of fibrosis in tissue (Wallace, Burt et al. 2008). Tissue sections were de-waxed and rehydrated as described in section 2.5.1. They were then immersed in picro- sirius red for 2-3 hours and then washed in 2 x 0.5% (v/v) acetic acid prepared in dH2O. Finally, sections were dehydrated and mounted as described in section

2.5.1. Sirius red staining was quantified by using the Leica software programme.

2.5.4 Double immunofluorescence staining

Tissue sections were de-waxed and rehydrated and antigen retrieval carried out as described in section 2.5.1. Tissue sections were then washed in 1 x PBS prior to blocking non-specific binding of antibodies with 20% FCS (v/v) in 1 x PBS for 30 minutes. Primary rabbit antibodies were diluted in 1 x PBS containing 0.05% (v/v) FCS (Table 2.7), added to each section and then incubated overnight at 4o_C.

Antibodies were added together if raised against different species for example, rabbit against target-1 and mouse against target 2. No primary antibody controls were incubated with blocking buffer alone. Following overnight incubation, sections were washed 2 x for 5 minutes in 1 x PBS before being incubated in the dark for 1 hour at room temperature with the appropriate flurochrome secondary antibody (Table 2.7). Sections were then counterstained with DAPI (4',6-

diamidino-2-phenylindole) for 10 minutes and then rinsed in 1 x PBS for 5

minutes. DAPI is a fluorescent probe that binds to AT regions of double stranded DNA, thus cell nuclei can be visualized. To quench auto-fluorescence, sections were immersed in Sudan Black (Sigma-Aldrich, Gillingham, UK) (0.3% (w/v) made up in 100% ethanol and filtered before use) and placed on a rocker for 30- 45 minutes. After rinsing in 1 x PBS 4 x 10 minutes, sections were mounted with a cover slip with a small drop of fluorescence mounting medium (Dako UK Ltd, Cambridgeshire, UK)and the cover slip edges sealed with nail varnish to prevent drying and movement under the microscope.

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Sections were observed under confocal microscopy using a Leica TCS SP II laser-scanning confocal microscope and LCS Lite 2.61 software. The basic principle behind confocal microscopy is that it uses point illumination in order to eliminate out-of focus signal. This is achieved by the pinhole being located in front of the detector thus, allowing only fluorescence to be detected when close to the focal plane.

2.5.5 Immunocytochemistry

For fluorescence microscopy, cells were grown in 6 well plates. The media was removed from the 6 well plates and washed twice in 1 x PBS. Cells were permeabilized with 100% methanol (Fisher Scientific, Loughborough, UK) for 10 minutes and fixed with fixation buffer (2% (v/v) formaldehyde, 0.2% (v/v) glutaraldehyde in PBS, pH7.4) for 20 minutes at -20o_{C. If cells were not stained}

immediately they were stored with 2mls of 1 x PBS in each well at 4o_{C. For}

staining, cells were blocked with 2mls of 20% FCS (v/v) in 1 x PBS for 30 minutes. Cells were then incubated in the presence or absence of a primary antibody (Table 2.7) at room temperature for 1 hour or overnight at 4o_{C. After incubation,}

cells were washed 2 x 5 minutes with 1 x PBS with gentle agitation and detected with the appropriate secondary antibody (Table 2.7) in the dark to prevent quenching of fluorescence signal. They were then washed a further 4 times in 1 x PBS for 5 minutes each. The PBS was removed and cells were counterstained with DAPI for 10 minutes, the cells were washed in 1 x PBS for 2 x 5 minutes and images were captured by fluorescence microscopy.