Tissue Microarray
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How to Prepare Tissue
Before You Begin
Please contact the TPSR and/or Dr. Katherine Gibson-Corley (katherine.gibson-corley@vumc.org) before you begin harvesting your tissue specimens. We are happy to consult on experimental design and tissue collection for optimal and timely results. We can also provide you with 10% neutral buffered formalin (10% NBF), the correct cassettes for your specimens and other supplies you may need to prepare your samples.
Fixation
Inadequate fixation affects every stage of tissue preparation from processing, sectioning, special staining to immunohistochemistry. The biggest problem we encounter in the laboratory is inadequately fixed tissues.
Proper fixation is the most important step of the entire process. Improper fixation cannot be corrected at any later stage of processing or staining.
The goal of fixation is to stop autolysis. A fixative must be able to penetrate tissue specimens as quickly as possible to prevent post-mortem changes. Several factors affect the rate of fixation including time, thickness of the tissue, temperature, volume of fixative to tissue, and type of fixation solution.
Time
It is more important to be sure tissue specimens are fixed long enough. Underfixation is the most common problem we see. As a general rule of thumb tissues should be fixed in 10% neutral buffered formalin overnight prior to delivery to the laboratory. Tissues should be delivered to the lab in 10% NBF.
Thickness of Tissue
The larger the piece of tissue is, the longer it will take to be completely fixed. The goal of fixation is to fix tissue as quickly as possible. Fixation proceeds from outside to inside, so cells near the surface will fix sooner than cells on the interior of the specimen. Smaller pieces of tissue will fix faster than larger pieces. Organs that are encapsulated will need to be cut in half to allow the fixative to penetrate to the center of the tissue.
IMPORTANT: Tissues placed in cassettes prior to fixation must be a maximum of 3mm thick.
Temperature
Room temperature is ideal for fixation. Placing specimens in the refrigerator slows down the rate of fixation which can result in suboptimal staining.
Fixative to Tissue Ratio
As a general rule of thumb, specimens should be placed into 10 times the volume of fixative to tissue. If the specimens are very large or need several days to fix properly the specimens should be placed into fresh fixative every day.
Type of Fixative Solution
Not all fixatives are alike. The best example of this is 10% neutral buffered formalin and 4% paraformaldehyde. These two solutions are not the same in composition or rate of fixation. 10% neutral buffered formalin will cross-link the proteins in specimens more quickly than 4% paraformaldehyde. 10% formalin is very rarely made up in labs, is readily available from most scientific vendors, comes ready to use (no dilution is required), and is stable for long periods of time at room temperature. Paraformaldehyde is purchased in powder form and must be diluted to 4% in PBS. 4% paraformaldehyde should be made up just before use because of its poor shelf life and must remain refrigerated. We highly encourage the use of 10% neutral buffered formalin for fixation. 4% paraformaldehde gained widespread useage when investigators were performing a lot of in situ hybridization work. Unless you are actively using these procedures, please consider using 10% neutral buffered formalin.
Cassette Labeling
All tissue delivered to the laboratory must be in pre-printed tissue cassettes. We will not accept tissue that have not been placed in cassettes. Please contact TPSR-Comparative_Pathology@vumc.org to order pre-printed cassettes.
Pre-printed cassettes have two lines with 9 characters available on each line.
For large cassettes that can not be printed, always use a pencil for cassette labeling. Ink from sharpies, markers, and pens WILL WASH AWAY in the alcohol and xylene utilized during the tissue processing procedure.
If we cannot decipher tiny or illegible handwriting we will hold your workorder until we can get in touch with you to properly identify the specimen.
Frozen Specimen Preparation
Samples can either be snap-frozen in liquid nitrogen and placed directly into aluminum foil or they can be placed in cryomolds and embedded and frozen in OCT. Please wrap cryomolds in aluminum foil also. Neatly label both the cryomold and the outside of the aluminum foil. Please bring to us on ice so they will remain frozen during the transport to the Core.
If you have any question about how to prepare your tissue specimens for either paraffin sectioning or frozen sectioning please contact TPSR-Research_Histology@vumc.org for assistance.
Routine Histology
Our full service research histology laboratory offers paraffin embedding, sectioning, automated Hematoxylin and Eosin staining, frozen sectioning and a large selection of special stains. The laboratory also offers specialized processing and sectioning services including RNase free sectioning, special processing for lacZ stained samples, and tissue microarray sectioning. The lab is continually developing new protocols to meet the special needs of requesting investigators.
The facility also offers access to a laser capture microdissection system and tissue microarray equipment for making microarray blocks.
We accept and process tissues prepared using the following methods:
- Fixed tissue submitted in 10% formalin, 4% paraformaldehyde, 70% alcohol, Bouins solution, or Davidson's solution
- Fresh tissue for frozen sections. Instructions for freezing methods and ordering of supplies can be arranged by contacting us at TPSR-Research_Histology@vumc.org.
- Embedded frozen tissue
- Tissue for decalcification (structures containing bone or cartilage)
For special fixation processes or procedures, please contact the Laboratory at TPSR-Research_Histology@vumc.org prior to tissue submission.
Sectioning
- Tissues are routinely sectioned into 5-μm sections, unless specified otherwise on the Histology Laboratory Request Form (This applies to both paraffin-embedded and frozen tissue blocks)
- Serial sectioning of blocks
- Step sectioning
Special Staining
All tissue blocks are routinely cut and stained with hematoxylin and eosin (H&E).
A wide variety of special stains are available. For less common stains, contact Laboratory at TPSR-Research_Histology@vumc.org to check their availability.
Personalized Service
Personalized services are available by reservation. Time can be reserved with a histologist to develop unique protocols or with a technician to identify structures of special interest while the tissue is being sectioned.
Special Stains
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Acid-Fast Bacteria (AFB)
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Luxol Fast Blue-Cresyl Etch Violet
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Alcian Blue, pH 2.5
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Movat’s Pentachrome
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Alcian Blue/PAS
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Oil Red O (frozen tissue only)
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Alizarin Red
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Periodic-Acid Schiff (PAS)
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Bielschowsky
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Von Kossa
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Congo Red
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Picrosirius Red
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Elastic
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Giemsa
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Safranin O
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Gram Stain
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Toluidine Blue for mast cells
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Grocott’s Methenamine Silver (GMS)
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Trichrome Blue
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Iron Stain
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Trichrome Green
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Immunohistochemistry
The following antibodies are currently in stock and are available on demand
| Mouse | Human |
| ACE2 | ACE2 |
| BrdU | Bcl-2 |
| Caspase-3 | BrdU |
| CD3 | Caspase-3 |
| CD4 | CD3 |
| CD8 | CD4 |
| CD11b | CD8 |
| CD31 | CD20 |
| CD34 | CD31 |
| CD45R (B220) | CD34 |
| CD45lca | CD45lca |
| CD56 | |
| Cyclin D1 | CD68 |
| Cytokeratin (AE1/AE3) | CD274 (PD-L1) |
| E-Cadherin | COX2 |
| ER | Cytokeratin (AE1/AE3) |
| Factor VIII (vWF) | E-Cadherin |
| Fox p3 | ER |
| F4/80 | Factor VIII (vWF) |
| GFAP | Fox p3 |
| Glucagon | GFAP |
| Iba1 | Iba1 |
| Insulin | Glucagon |
| Ki67 | Insulin |
| LY6G | Ki67 |
| Major Base Protein | Melan A |
| Myeloperoxidase | Myeloperoxidase |
| Neutrophil Marker | PDCD1 (PD-1) |
| PR | PR |
| SMA | SMA |
| S100 | S100 |
| TUNEL | TUNEL |
| Vimentin | Vimentin |
Antibody optimization and validation
We have also validated/optimized a wide range of antibodies that we do not keep stocked in the lab. These antibodies will need to be purchased by the client. Please contact TPSR-IHC@vumc.org for more information on antibodies of interest not included in our on demand list.
Antibody titration and dilution studies can be performed on your antisera of interest.
Please inquire.
Results cannot be guaranteed if samples are not processed or sectioned in the TPSR.
Tissue Microarray
Tissue microarray instruments allow generation of multiple specimen slides that contain hundreds of individual tissues. Instead of incubating and analyzing samples one slide at a time, tissue microarrays (TMAs) allow the investigator to examine hundreds of samples with just one slide.
Tissue microarray (TMA) construction and biomarker staining is performed by the Translational Pathology Shared Resource. The process involves three steps that include, consultation, construction, and biomarker staining. During the consultation phase, investigators meet with the TPSR staff to select appropriate core size, identify blocks and slides, and design the TMA map. Once the TMA map design is approved by the investigator and blocks and slides are organized, the construction phase begins. Tissue blocks are loaded on to the TMA Grandmaster (PerkinElmer) automated arrayer and the TMA map is loaded into the system and the automated construction begins. Following construction, an H&E slide is made from the TMA and evaluated for quality. Once the slide is approved, additional slides are cut and biomarker staining is performed.