Medicine

Microscopy

The microscopy core facility provides a specialist microscopy service to the Division as well as providing training and advice to staff and students in microscopy techniques.

All the resources necessary to carry out immunohistochemistry and immunofluorescence microscopy studies on frozen tissue or cell cultures as well as post-embedding processing of paraffin embedded tissue and live cell confocal microscopy are provided.

Staff: Dr Sonya James (nee Martin); Part time, Wed-Fri


Services provided

  • Freezing tissue samples and cryosectioning
  • Immunolabelling of tissue sections or cells
  • Live cell confocal imaging
  • Image collection and analysis.
  • Preparation of figures to publication standard
  • Training in microscopy techniques, advising on the planning of experiments and assisting with image collection and analysis
  • Providing information on the facilities available within The Faculty and helping with the planning of microscopy related projects
  • The facility enjoys a close working relationship with The Faculty of Medicine, Biomedical Imaging Unit (BIU) and
  • Histochemistry Research Unit (HRU).

 

Equipment provided

Microscope
Olympus CKX41 inverted microscope for bright-field and phase contrast and fluorescence imaging complete with digital camera and PC to enable straightforward image acquisition of cells in culture or cells/tissues labelled for light microscopy. The microscope is equipped with blue, red and green filters allowing the collection of UV (eg Dapi), green (eg FITC) and red (eg TRITC) fluorescence, respectively. Currently the microscope is equipped with x4, x10, x10 (phase contrast) and x40 objective lenses. A x20 fluorite lens is also available on request.

Microm H500 cryostat - for cutting frozen tissue sections

Cytocentrifuge - for preparation of cytospins

All other microscopy is carried out at the BIU which has two confocal microscopes (both laser scanning); a Leica SP2 for the acquisition of 2-4 colour fluorescence and a Leica SP5 capable of up to 5-colour fluorescence imaging and live cell work.

Costs

Individual groups may have to purchase specific primary antibodies or reagents specific for their particular study. All other general reagents are supplied.

Fluorescence and confocal microscopes provided by BIU are charged at an hourly rate. (www.biu.soton.ac.uk)

Paraffin embedding and routine H&E staining of paraffin sections is carried out by HRU charged on a per block or per section basis. (http://www.hru.soton.ac.uk/)


Please do not hesitate to contact me if you have any queries.

Dr Sonya James
Microscopy Support Officer
Cancer Sciences Division
Tenovus Research Laboratory
G6 and 7, Ground Floor
Tel: 02380 796589
Email: sonya.james@soton.ac.uk

 

Examples of work carried out by the Microscopy Facility

Immunofluorescence (frozen sections)

See Fig 1

Depletion of B-cell subsets in the lymphoid compartment.

As published in; Beers SA et al (2008) Blood 112(10); 4170 http://bloodjournal.hematologylibrary.org/cgi/content/full/112/10/4170

See Fig 2

Administration of aGalCer triggers CD70 on DCs in the T cell areas of the spleen

As published in; Taraban VY et al (2008) The Journal of Immunology 180; 4615 http://www.jimmunol.org/cgi/content/full/180/7/4615?maxtoshow=&hits=10&RESULTFORMAT=&author1=taraban&searchid=1&FIRSTINDEX=0&sortspec=relevance&volume=180&resourcetype=HWCIT

Immunohistochemistry (frozen sections)

See Fig 3

Immunohistochemical analysis of mAb distribution in the spleen after i.v. injection. 

As published in; Castro FVV (2008) Eur. J. Immunol. 38; 2263 http://www3.interscience.wiley.com/cgi-bin/fulltext/120846837/HTMLSTART

Immunofluorescence (cultured cells)

See Fig 4

Human ERAAP can be detected in ER but not the Golgi after transfection into 293T

Recent Publications

White AL, Tutt AL, James S, Wilkinson K, Castro FVV, Dixon SV, Hitchcok J, Khan M, Al-Shamkani A, Cunningham AF and Glennie MJ (2010) Ligation of CD11c during vaccination promotes germinal centre induction and robust humoral responses without adjuvant. Immunology 130(2):

Gray JC, French RR, James S, Al-Shamkhani A, Johnson PW, Glennie MJ (2008) Optimising anti-tumour CD8 T-cell responses using combinations of immunomodulatory antibodies. Eur. J. Immunol. 38: 2499-2511

Castro FVV, Tutt AL, White AL, Teeling JL, James S, French RR and Glennie MJ (2008) CD11c provides an effective immunotarget for the generation of both CD4 and CD8 T cell responses. Eur. J. Immunol. 38: 2263-2273

Beers SA, Chan CHT, James S, French RR, Attfield KE, Brennan CM, Ahuja A, Shlomchik MJ, Cragg MS, Glennie MJ (2008) Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood 112 (10): 4170-4177

Taraban VY, Martin S, Attfield KE, Glennie MJ, Elliott T, Elewaut D, Van Calenbergh S, Linclau B and Al-Shamkhani A (2008) Invariant NKT Cells Promote CD8-Cytotoxic T Cell Responses by Inducing CD70 Expression on Dendritic Cells. The Journal of Immunology 180: 4615-4620.

Fig 1

Fig 1

Fig 2

Fig 2

Fig 3

Fig 3

Fig 4

Fig 4

Three colour labelling

of cultured cells

Three colour labelling

Three colour labelling

of mouse spleen

Three colour labelling

Neutrophils (yellow)

and macrophages (blue) in mouse spleen

Neutrophils (yellow)

T cell infiltration of murine

pancreatic islet (Immunohostochemistry, frozen section, Light microscopy)

T cell infiltration of murine