- Preparation of Paraffin Sections:
- Paraffin Processing
- Paraffin Embedding
- Paraffin Sectioning
- Preparation of Frozen Sections:
- Embedding for Cryosectioning
- Cryostat sectioning
- Cryostat Muscle Sectioning
- Preparation of Plastic Sections:
- Glycol Methacrylate (GMA) Embedding
- GMA Sectioning
- H&E Staining
- Special Stains
- X-Gal Staining
- Alkaline Phosphatase Staining
- In Situ Hybridization:
- Probe Preparation
- TUNEL Assay
- Electron Microscopy (EM):
- EM Processing/Embedding
- EM Sectioning/Staining
- Regular (Full) EM
- EM Negative Staining
- Grossing In
Preparation of Paraffin Sections
Tissue processing cassettes are labeled with the CMC #, study #, study day, animal # or any other designation listed on the container or service request form.
Areas of interest from fixed tissue pieces are transferred into labeled cassettes and placed in the automatic tissue processor. Processing starts in the evening and ends the next morning.
The paraffin blocks are trimmed, mounted in the microtome, and serial sections are cut from each block.
Preparation of Frozen Sections
Embedding for Cryosectioning
All specimens are placed in molds of appropriate size that allow approximately 0.5 cm of space between the edge of the tissue and the side of the mold. The surface to be sectioned is placed on the bottom of the mold, OCT is added, and the mold is immersed in isopentane cooled with liquid nitrogen.
Frozen tissue is mounted in the cryostat for cutting individual sections in the 6-10 Ám range.
Cryostat Muscle Sectioning
Muscle tissue is frozen onto pieces of cork without submersion into OCT medium to guarantee maximal freezing speed.
Preparation of Plastic Sections
Glycol Methacrylate (GMA) Embedding
Fixed tissue specimens are dehydrated through an ethanol series and then embedded in capsules filled with GMA. For thin sections, 1 Ám sections are obtained and stained with H&E or Toluidine Blue.
Glass knives are used to section GMA-embedded tissues at 1 Ám.
The standard staining technique requires the use of hematoxylin and eosin (H&E).
Routine hematoxylin and eosin staining is by far the most commonly requested stain to evaluate histopathologic changes. However, additional special stains can be helpful in detecting subtle alterations in tissue and organ architecture. Tissue scarring can be detected by trichrome in that areas of fibrosis are accentuated and reticulin staining will demonstrate areas where there is an excess deposit of reticulin. Periodic acid Schiff staining will detect various glycoproteins and oil-red-O staining is done to identify various lipid deposits such as microvesicular steatosis in the liver.
The histochemical detection of beta-galactosidase ("X-Gal" staining), commonly used as reporter for transgene expression, is a frequently requested service and can be performed on tissues and cells.
Alkaline Phosphatase Staining
Human placental alkaline phosphatase (hPLAP) is also used as a reporter to assess transduction efficiency. To detect hPLAP expression, tissues are heat-treated to inactivate endogenous phosphatases and stained with NBT/BCIP.
In Situ Hybridization
The Cell Morphology Core is using digoxigenin-labeled probes for in situ hybridization which are detected either with fluorescent or alkaline phosphatase-labeled antibodies. If possible, investigators should provide plasmids containing the gene of interest suitable for in vitro transcription (i.e. with T3, T7, or SP6 promoters) to allow the preparation of RNA probes (riboprobes). Alternatively, DNA probes can be prepared from any template either by nick translation or PCR. If no template DNA is available, oligos can be designed and labeled by terminal transferase with an digoxigenin "tail". Usually a mix of several oligos is used for the detection of one RNA species to enhance sensitivity.
Frozen as well as paraffin sections can be used for fluorescence or chromogenic in situ hybridization. We perform ISH both for DNA and RNA targets in cells or tissue sections. Hybridizations are always run with the appropriate controls, including the use of a sense probe in case of RNA probes or unspecific sequences for other types of probes.
Specimens to be examined by immunostaining may include cells (adherent cells or suspension cells spun down on slides), cryostat sections, or paraffin sections. Both fluorescent or enzyme labeled antibodies (usually peroxidase with DAB as substrate) can be used for the immunological detection of proteins. The CMC has an automated immunostainer (Ventana) which can process up to 120 slides simultaneously. This immunostainer uses capillary gap action to draw reagents onto the slide surface and blotting pads to withdraw the reagents. Once optimal protocols have been developed at the lab bench, the automated station is programmed to perform the same protocol for large scale staining operations.
The TUNEL assay detects degraded DNA as an indicator for apoptotic events. Tissues treated with DNase are used as positive controls. Alternatively, immunostaining against apoptosis-related proteins can be performed.
Electron Microscopy (EM)
Tissue specimens are minced with a razor blade and fixed in glutaraldehyde and osmium tetroxide. Specimens are dehydrated in graded alcohols and embedded in capsules filled with Epon which is polymerized in an 70 °C oven for several days.
EM Sectioning/Staining (includes microscopy and printing images)
Serial sections are cut on an ultramicrotome and collected in a "boat" filled with filtered, distilled water. For standard electron microscopy tissue sections of 80 nm are prepared, stained with lead citrate, and images are taken with a Philips CM-100 transmission electron microscope.
Regular (Full) EM
This full service includes the whole process of electron microscopy: fixation of the tissue, processing and embedding, sectioning, taking pictures and printing images.
EM Negative Staining
Negative staining is used at the CMC primarily to evaluate viral vector preparations (adenovirus and adeno-associated virus), but can also be applied to examine other small particles such as DNA conjugates of nonviral vectors. For each evaluation, a minimum of 5 Ál virus suspension is needed. Capsid integrity, purity of the preparation, clumping of viral particles, and contamination with other viruses are examined for each preparation.
Pre- and post-embedding techniques can be performed for immunostaining at the electron microscope level with gold-labeled antibodies. Immunostaining is performed either before or after embedding the sample for EM. For tissues, usually post-embedding techniques are used with Lowicryl K4M as resin which allows antibodies to access the tissue section.
CMC performs necropsies on animals of all sizes and collects tissues according to their future use.
This process involves the trimming of organ specimens taken at necropsy prior to fixation and embedding
Prepared slides can be submitted to a pathologist and interpretations concerning tissue structure and pathology are given. Interpretation of special stains and immunohistochemistry are also provided