.

Translational Pathology Shared Resource

TPSR

Comparative Pathology
Phone: 615.322.3667
Room: MCN S1310

Human Tissue Acquisition
Phone: 615.343.3274
Room: TVC 4902

Immunohistochemistry
Phone: 615.322.3667
Room: MCN S1310

Research Histology
Phone: 615.322.3667
Room: MCN S1310

Research Histology

   

 

Routine Histology

Special Stains

Immunohistochemistry

Laser Capture Microdissection

Tissue Microarray

How to Prepare Tissue

____________________________________________________________________________________

 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 cell block preparations, 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 or 4% paraformaldehyde
  • Fresh tissue for frozen sections.  Instructions for freezing methods and ordering of supplies can be arranged by contacting us at TPSR-Research_Histology@vanderbilt.edu.
  • 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@vanderbilt.edu 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@vanderbilt.edu 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

 

Acid-Fast Bacteria (AFB)
Luxol Fast Blue-Cresyl Etch Violet
Alcian Blue, pH 2.5
Movat’s Pentachrome
Alcian Blue/PAS
Oil Red O (frozen tissue only)
Alizarin Red
Periodic-Acid Schiff (PAS)
Colloidal Iron
Phosphotungstic Acid Hematoxylin
Congo Red
Picrosirius Red
Elastic
Reticulin
Giemsa
Safranin O
Gram Stain
Toluidine Blue for mast cells
Grocott’s Methenamine Silver (GMS)
Trichrome Blue
Iron Stain
Trichrome Green
Jones’ stain for basement membranes
Warthin-Starry
Luna’s stain for eosinophils
 

__________________________________________________________________________________

Immunohistochemistry

The following antibodies are currently in stock and are available on demand (m = mouse; h = human):

 

 

Immunophenotyping
Angiogenesis
Other markers
CD3 (m)
CD31 (h) (m)
BRCA1 (h)
CD4 (h only))
CD34 (m)
CD274 (PD-L1) (h)
CD8 (h only)
Factor VIII (vWF) (h) (m)
COX2 (h)
CD8 (m-frozen only)
Proliferation
Cyclin D1 (m)
CD11b (m)
Ki67 (h)  (m)
Cytokeratin (h) (m)
CD45R (B220) (m)
PCNA (h) (m)
E-Cadherin (m)
F4/80 (m)
BrdU (m)
GFAP (m)
Fox p3 (m)
pHistone-H3 (m)
Glucagon (m)
Major Basic Protien (m)
Apoptosis
Insulin (m)
Myeloperoxidase (h) (m)
Caspase-3 (h) (m)
Melan A (h)
Neutrophil Marker (m)
Extracellular Matrix
p16 (h)
PNA (m)
Laminin (m)
PDCD1 (PD-1) (h)
Ter119 (m)
Collagen IV (m)
S100 (h) (m)
 
Fibronectin (m)
Vimentin (h) (m)

 

 

 

 

 

 

 

 

 

 

 

 Antibody optimization and validation

We have also worked with a wide range of antibodies we do not keep stocked in the lab. These antibodies will need to be ordered by our clients. Please contact TPSR-Research_Histology@vanderbilt.edu for more information on any of the antibodies below or any other antisera of interest (m = mouse; h = human)

 

 

 

Aldh2 (h)
Galectin-3 (h)
Olig-2 (m)
ALOX5AP (h)
Gelsolin (h)
p44/42 MAPK (h)
Androgen Receptor (m)
GLP1R (m)
p53 (h)
B-Catenin (h) (m)
GSK-3B (m)
PAI-1 (m)
Bcl-2 (h)
Hemoglobin A (h)
Pan Actin (h)
Calcitonin (h)
HNF-1 Beta (h)
Parvalbumin (m)
Calcyclin (h)
HSP90B1 (h)
PAX8 (h)
CD10 (h)
ICAM1 (h)
Paxillin (h)
CD206 (m)
IGF1 (m)
pGSK-3B (m)
CD53 (h)
IGF2 (h)
pH2Ax (h)
CD56 (NCAM1) (h)
IkBa (h)
phospho-Smad2 (m)
CD62E (E-selectin) (h)
IMP3 (h)
phospho-Stat3 (h)
CD62P (P-selectin) (h)
KEAP1 (h)
PKC alpha (h)
c-myc (h)
Ku p80 (h)
PR (h)
Cyclophilin A (m)
Ly6G (m)
RAGE (m)
DEFA5 (h) (m)
LYN (h)
SAA1 (h)
Desmin (h)
LZAP (h) (m)
SCF (m)
DKC1 (h)
MASH1 (h)
SLC1A5 (h) (m)
DR4 (m)
Melanosome (h)
Substance P (h)
eNOS (h) (m)
MMP9 (r)
TGF beta (m)
Foxa1 (m)
MMP-12 (m)
Thymosin B10 (h)
Foxa2 (m)
MMP-14 (m)
UCP1 (m)
FSP1 (m)
NF-kB p65 (h)
VCAM1 (h)
FYN (h)
Nrf2 (h)
VEGF (h)
Galectin 7 (h)
Nucleophosmin (h)
Wip1 (h) (m)

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.

 

 

TPSR Immunohistochemistry Request Forms

TPSR Workorder Form 

__________________________________________________________________________________

Laser Capture Microdissection

 

Laser Capture Microdissection (LCM) systems rapidly isolates pure cell populations for cell-specific analysis and enables the investigator to increase the sensitivity and accuracy of molecular assays by starting with samples of homogeneous cell types and multi-cellular structures isolated from whole tissue or cytology samples.  LCM has enormous flexibility with respect to tissue and cell fixation preparations.  LCM effectively extracts cells from both paraffin-embedded and frozen tissue sections prepared using a wide variety of different dyes, slide surfaces, and protocols, providing quality material for a wide variety of DNA, RNA, and protein analyses.

__________________________________________________________________________________

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. 

__________________________________________________________________________________

How to Prepare Tissue

 

Before You Begin

 

Please visit the TPSR before you begin harvesting your tissue specimens.  We can 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. 

 

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 tissue cassettes.  We will not accept tissue that has not been placed in cassettes.  

 

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.

 

We are unable to print symbols or labels over 12 characters in length on our slides.  We suggest you consider using simple accession numbers (such as 10-50A, 10-50B, etc. or 2010-50A, 2010-50B).

 

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@vanderbilt.edu for assistance.

 

 

 

 

 

 

 

 

This page was last updated September 17, 2012 and is maintained by