Core C - Cell Imaging and Tissue Histology
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This Core provides DDC investigators with resources and support in rthe following areas:
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Conventional light and fluorescence microscopy
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Laser scanning confocal microscopy
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Laser capture microdissection
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Tissue histology service and training
The Core's components are located in the Division of Gastroenterology & Hepatology's laboratories at Stanford University's Alway Building, Rooms M203 and M207, and at the Palo Alto VA research facility in Building 101, fourth floor, Rooms C4-120, C4-131 and F4-211A. The VA research facility is located approximately 3.7 miles away from Stanford and travel between the two institutions takes approximately 11 minutes by car.
New equipment obtained in the last funding segment and located at the Palo Alto VA include two new Zeiss laser scanning confocal microscopes, a new Zeiss LSM71a Axioimager epifluorescent microscope, a Leica LMD 6000 laser capture microdissection microscope, and a new Leica cryostat. On the Stanford campus, the Stanford DDC has added a new Nikon laser scanning confocal microscope and a new Leica cryostat.
Questions concerning the use of services can be directed to Evelyn Resurreccion at the Palo Alto VA, or Aparna Gupta at Stanford. You may also contact the Core Director, Anson Lowe, or the DDC Administrator, Linda Jacob.
Standard light and fluorescence microscopy
A total of three standard light and fluorescence microscopes are available for use by the DDC membership. Two were acquired by the Gastroenterology & Hepatology Division and DDC and are available on Stanford campus. The Nikon Eclipse E600 is located in the Gastroenterology & Hepatology Division laboratories at Alway Building (Rm. 205), and the inverted Nikon Eclipse TE300 microscope is at the CCSR Building (Rm. 3110). A third epifluorescence microscope, a Zeiss Axioimager D1, is located at the Palo Alto VA (Building 101, Rm. C4-130).
The availability of these microscopes provides several advantages. First, evaluation for most labeled samples is much faster and more efficient on standard epifluorescence microscopes. Thus, many investigators choose to perform evaluation on a standard microscope before deciding to use the confocal microscopes. Second, the Nikon E600 microscope at Stanford is equipped with an RT Spot digital camera that is capable of color images. This enables evaluation and documentation of images using standard H&E and peroxidase immunohistochemical stains.
Laser scanning confocal microscopy
Laser scanning confocal microscopy provides several advantages over traditional fluorescence microscopy. The images appear much sharper because the out-of-focus background is excluded. This feature is especially helpful when examining thick specimens. Additionally, the confocal microscope is able to restrict signal input to a specific focal plane and the microscopists can literally "section" their way through the specimen without actually cutting and destroying the tissue.
With respect to techincal support, Ms. Evelyn Resurreccin continues to support immuno-histochemistry services and confocal microscopy training at the Palo Alto VA. We also have added Dr. Aparna Gupta to manage the microscopy resources at Stanford.
Confocal at the Palo Alto VA
Confocal microscope, Stanford
Since the last funding period, the DDC has acquired three new laser scanning confocal microscopes. Located at the Palo Alto VA are two microscopes, namely a Zeiss LSM 510 Meta and a Zeiss LSM710. The LSM710 features : 1) 8 laser lines; 2) continuous spectral detection for up to 10 dyes at once without any crosstalk; 3) improved PMTs with higher sensitivity and signal to noise rartios; 4) Multiphoton capabilities. All interested users should contact Evelyn Resurreccion at the VA to arrange a training.
The Digestive Disease Center at the Stanford campus has recently acquired a Nikon confocal microscope that will supplement the Bio-Rad MRC 1024 confocal. The microscope is housed in the Division of Gastroenterology located in the Alway Building, room M207 on the Stanford Campus. The new confocal unit features a Nikon TS-1 inverted microscope equipped with long working range lenses from 4X, 10X, 20X, and 40 X., which enables imaging of live cells in tissue culture plates or chambers. The microscope also includes high-resolution short working range lenses of 20 X, 40 X, and 60 X (oil) that enable high-resolution imaging of specimens mounted on microscope slides. The microscope is equipped with three lasers that enable three color fluorescence.. The microscope is also equipped with a Z-focus drive that enables automatic software driven image acquisition in a Z-stack format. DDC members interested in using the Nikon confocal at Stanford should contact Aparna Gupta by email or telephone for a training session and clearance to use the microscope. A step-by-step guide on how to use the microscope is available here as a PDF file. For protocols on using the microscope, please click on the link below.
Core C PDF 110909Laser capture microdissection
DDC members have access to a recently acquired Leica AS LMD. The LMD instrument, located at the Palo Alto VA (Rm. A4-121) consists of a Leica DM6000B microscope that is attached to a Leica LMD 6000 laser capture unit. The equpment features motorized focusing and stage movement. It also features fast automated laser cutting that can obtain single cells for genomic or proteomic analysis. DDC members interested in using the microscope should first contact Evelyn Resurreccion for training and clearance to use the instrument.
Tissue histology
The DDC provides support for services for the preparation of histological samples and the development of protocols for specific samples. This service is offered as a means to complement the increasing use of genetically altered mice and human specimens. Although numerous protocols exist for the preparation of samples, there is no uniform approach. Each type of tissue and each individual antibody often require an empirical assessment of multiple protocols with the addition of slight modifications. In these cases, the experience and expertise possessed by the DDC's histology staff greatly enhance productivity.
The Tissue Histology subcore is composed of two components that encompass both services and training:
a.Tissue preparation and staining services. The DDC has engaged the services of the histology lab located in the Department of Comparative Medicine in the Stanford Edwards Building (Rm. 330), adjacent to the Division of Gastroenterology & Hepatology. Under the direction of an experienced histology technician, Ms. Pauline Chu, the histology lab is a service center that provides a comprehensive range of techniques that include standard paraffin processing and embedding methods as well as hand processing for larger specimens; special orientation of tissues; serial sectioning; step sectioning; and sectioning of frozen specimens. DDC members are provided discounted rate for use of these services. Interested members should first contact the DDC Administrator, Linda Jacob, at email lpjacob@stanford.edu or telephone no. 650-724-6469 or 650-493-5000 x 63349.
b. Frozen crysections. Fewer than 20% of antibodies are able to specifically bind antigens that are fixed and embedded in paraffin. As a result, frozen cryosections must be used as they often result in better entigen preservation. The DDC provides the resources and training for frozen cryosections. The DDC supports Ms. Resurreccion to teach DDC members how to cut frozen sections. Once the users obtain sufficient proficiency, cryostats are available for use. A new Leica CM-3050S Cryostat was purchased by the DDC and is available at the Palo Alto VA. A second new cryostat (Leica CM-1905) was purchased and is located in the GI Division space at Stanford University (Alway building, Rm. M215).
 Microscope training. Evelyn Resurreccion provides training with the laser confocal microscope and cryostat. |
 Confocal Images. Confocal immunofluorescence three-dimensional reconstructions of MDCK monolayers infected with H. pylori for 4 hours ( A ) or 8 hours ( B and C ), and stained with antibodies to ZO-1 (green), E-cadherin (blue), and H. pylori (red) are shown. Yellow areas indicate spatial overlap (colocalization) of the red-stained bacteria with the green ZO-1 signal. Arrows show ZO-1 recruited to extrajunctional sites of bacterial attachment in MDCK cells. Scale bar, 10 µm in (A) and 5 µm in (B) and (C). Science 300: p1430 (2003). Performed by DDC RBIs Manuel Amieva, Stanley Falkow, and W. James Nelson. |
