Services

Tissue Preparation/Embedding/Sectioning: The primary services offered will be: 1) fixation and cryosectioning preparatory for immunocytochemistry, and 2) fixation, resin embedding and sectioning for high resolution LM and EM.  Ultracyromicrotomy and embedding in special resins such as Lowicryl or LR White can also be performed.  Fixation procedures will be discussed with the investigator. Instruction can be given in perfusion fixation of animals or fixation of cell subfractions in ultracentrifuge tubes.  Fixatives and their recipes are available from the Core or alternatively tissue is given to the Core for fixation.  Paraffin embedding will not be performed and investigators requiring this service will be directed to the Department of Pathology.  Samples for resin embedding are processed with osmication as needed, dehydrated and embedded usually in Epon resin.  Each sample is sectioned as 1um sections, stained with toluidine blue and evaluated on the light level prior to electron microscopy.

Transmission Electron Microscopy:  Blocks prepared as above will be sectioned as 60-100 nm ultrathin sections and picked up on nickel or copper grids.  For routine analysis, these will be stained with uranyl acetate and lead citrate and viewed and photographed on the Philips EM in the Department of Cell and Developmental Biology’s central research facility, the Microscopy and Image Analysis Laboratory (MIL).  Investigators are invited to be present when specimens are viewed.  Normally 5-10 images will be digitally captured and will be discussed with the Investigator.  When appropriate, publication quality photographs will be prepared including digital processing.  Immunocytochemistry by immunogold labeling or Protein A Gold will be carried out on cell fractions prior to embedding and for intact cells by on grid staining.  Unless the investigator has a tried and true protocol, this is labor intensive and requires trials with a number of fixatives, resins, and dilutions of antibodies.

Light Microscopic and Confocal Immunofluorescence Analysis:  The emphasis here is on confocal microscopy as the Core’s experience has been that this will improve upon almost every type of immunohistochemistry, and provides ready access to digital image analyses, including 3-D reconstructions.  Following conventional immunofluorescence to validate staining, sections will be analyzed on the LSCM either by Core personnel, or more generally, by users after training.  All users will be provided four free hours of instruction.  After this, involvement of Core personnel will be recharged.  It is anticipated that most users will want to capture their own images but some clinical investigators may want full service with provision of hard copy images.  Once trained, a user will sign up and use the microscope and store their own images on either a CD or DVD which can be provided by the Core at cost.  Images can also be transferred to the users’ computer via the network or portable USB hard drives.

Fluorescence Analysis of Living Cells: Initial consultation with the Core Director will determine whether the study is better performed on the confocal or wide-field fluorescent microscope.  High resolution work, work on thick specimens or studies requiring high speed time resolution will be performed on the Olympus confocal.  We have experience to date using fura-2, fluo3, calcium green and Magfura dyes for Ca 2+, SBFI for Na +, BCECF for H+, SPQ for C1-, lucifer yellow and lissarhodamine as gap junction tracers, and CFP, GFP and YFP fluorescent proteins as markers for transfected cells.  Other probes are available for potential sensing, labeling of mitochondria and following endocytosis.  Most projects in living cells have a developmental component and require continued consultation with core personnel.  Both instruments can be set up to vary the frequency of image collection which affects both the size of data files and bleaching of probes.

FRET:  Because all FRET work requires a large developmental component, this is not offered as a routine service.  The Core will provide instrumentation and training in its use.  At present, the Core is able to monitor energy transfer from CFP to YFP.  Advice can be given on construction of tagged proteins but normally the investigator will prepare these.  Other types of FRET will require purchase of appropriate filters by the investigator or jointly with the core if there is general interest.  The MIAC continues to offer investigators the ability to collect and analyze FRET data from the wide-field fluorescent and confocal microscopes.  The confocal is used primarily to perform acceptor (YFP) bleach protocols to measure and analyze FRET data and the wide-field system is used to perform stoichiometric FRET analysis. 

Image Analysis:  Image analysis services include quantitative morphometric analysis of images and preparation of publication quality images and slides.  Investigators can use the Core PC workstations on an hourly recharge or this service can be provided by the core personnel.  Quantitative analysis of static images can be performed using MetaMorph or ImageJ software on a PC for analysis of area, size, shape, distance, and relative position of defined cellular and subcellular structures.  Relative volume densities can also be calculated by the point counting method of Weibel.  Particle counting can be performed and expressed per cell area or membrane length.  Quantitative analysis including 3D reconstruction, quantification of fluorescence intensity, etc., can also be performed with Volocity software and carried out on our high-end 64-bit PC workstation.  Preparation of publication quality figures and montages can be done on our PC workstations using Photoshop, Prism, Illustrator, or PowerPoint software.  Instruction in the use of these programs is available from the Lab Director or the Associate Director. 

In situ Hybridization: The Core offers expert knowledge and assistance in the preparation of samples and running of in situ hybridizations.  The Laboratory Director, Dr. Lentz, has extensive experience with a large variety of in situ hybridization techniques for whole-mounts, tissue sections, and cultured cells from adult and embryonic tissues (mouse, rat, and zebrafish).  Hybridization signals can be detected with either isotopic (35S, 33P) or non-isotopic methods (fluorescent and colorimetric).  Multiple signals can be detected with double in situ hybridizations (using digoxigenin and fluorescein tagged cRNA probes) or in situ hybridizations combined with immunohistochemical staining of proteins (both peroxidase- and fluorescent-based detections of antibodies).

Autoradiography:  Although not routinely available as a Core service, the Core Director has experience with both light and EM autoradiography of newly synthesized protein to follow the secretory pathway and of localizing radioiodinated hormones bound to receptor on target cells.  The latter can also be used in expression cloning of receptors.  Thus the Core can provide assistance to an investigator interested in this technique.