Services

Tissue Procurement and Preparation

One of the most important keys to a successful IHC project is proper tissue procurement and preservation techniques. The facility provides advice and written protocols for proper tissue procurement **(see protocols). Here are some points to be aware of when preparing your tissues:

Tissue procurement involves either fixation of the tissue (generally called "Fixed") or tissue can remain unfixed (generally called "Fresh")
Next, the tissue is **embedded in paraffin or cryogenically frozen in O.C.T.
The choice of paraffin or frozen has to do with the tissue type, the antigen you want to detect with IHC, and the information you wish to gain from the experiment
Freezing is a better method for antigen preservation in immunohistochemistry.
Formalin-fixed paraffin-embedded (FFPE) tissue remains today the medium of choice for most clinical and research studies because of the superior preservation of morphology.
A major limitation of routinely processed FFPE tissues for immunohistochemistry is that many potentially interesting antigens are altered during tissue fixation and processing. Depending upon the antigen of interest, the antigen retrieval method may be necessary in order to unmask the antigen and make it to be detectable.

As you can see there are many things to take into consideration in the preparation steps of your experiment. Please contact the facility if you have any questions regarding which method best suites your experiment.

Tissue Sectioning and H&E Staining

Once the tissue has been properly prepared and procured, frozen and paraffin sections can be cut from the tissue block.
Frozen tissue, cut on the cryostat, and paraffin tissue cut on the microtome are sectioned onto slides.
Observations regarding the tissue morphology are made after staining with hematoxylin and eosin.
Hematoxylin stains the nucleus of cells, and Eosin stains the cytoplasm.
This ensures tissue integrity and an intact target site prior to immunostaining.
If poor morphology and a disturbed target site are found, the facility may request that new tissues be prepared again.

Investigators can section their own tissues using the facility's cryostat and microtome at a reasonable hourly fee. You can get information on how to use the microtome and cryostat by contacting the facility.

Immunohistochemistry

The use of antibodies provides specific cellular detection and location of cells within tissues. We have developed immunohistochemistry staining panels on a variety of tissues within the:

Immune system (spleen, lymph nodes, tonsil, thymus),
Circulatory system (heart, artery and vein),
Nervous system (peripheral nerves, brain, trigeminal ganglion and retina)
Other major organs including skin, lung, liver, kidney, pancreas, breast, and prostate.

Up to 3 antibodies can be used on one tissue section providing useful multi-parameter analysis and co-localization information.
A list of antibodies successfully used on paraffin and frozen tissue sections is available from this web site.
The facility will perform the staining according to the client’s personal preference of the methods (enzymatic base or fluorescence) and the colors, if any.
To see some of the staining that we have done here for our clients, visit the photo gallery.
We are always eager to explore new antibodies and new techniques. If your antibody, tissue type, or species isn't present on our lists, we can help develop a staining protocol for it.

In Situ Hybridization

In situ hybridization is a method of localizing either mRNA within the cytoplasm or specific DNA sequences within the chromosomes of the nucleus by hybridizing the sequence of interest to a complimentary strand of a nucleotide probe.
For in situ hybridization one utilizes a probe (or complimentary sequence) to detect specific nucleotide sequences within cells and tissues. The sensitivity of the technique is such that threshold levels of detection are in the region of 10-20 copies of mRNA or DNA per cell.
This technique allows investigators to not only be able to detect proteins on or in the cell/tissue, but to detect the DNA/mRNA precursors possibly before the actual proteins are made.

Investigators can submit cytospins, frozen section or paraffin-embedded material for ISH assays.
The probes must be provided by the investigator.

Developmental Panel for New Antibodies

The facility provides developmental studies using antibodies that have not been previously used in immunohistochemistry studies. These studies include

The use of different post-sectioning fixatives,
Implementation of various antigen retrieval methods including

Microwave,
Heat/steam combinations,
Pressure cooker,
Temperature variation or
Various buffers.
Enzymatic pretreatment, and
Antibody titration.

Again, this is for antibodies that have not been previously used in IHC experiments. It is up to the client to begin the research to find whether or not the antibody of interest has been used in IHC.
For antibodies purchased from a commercial vendor, spec. sheets and technical data sheets should be presented to the facility, as well as any publication material.