Services
Histology
Comparative Pathology
- Complete necropsy and histological analysis:
- Full work-up includes gross examination, as well as body and organ weights
- Histopathological evaluation of all systems (≈40 selected sites)
- Consultation and evaluation of histopathology in fixed tissue specimens collected by investigators
- Consultation for gene expression studies at the cellular level in histologic slides (including immunohistochemistry, β gal)
- Morphometric analysis to quantify histologic changes
- Documentation including description, gross photography and microphotography
- Training in animal and pathology procedures
Immunohistochemistry
- Indirect HRP and direct immunofluorescence
- Custom- antibody provided by us or principle investigator
- Protocol development- Please discuss with us before purchasing antibodies if possible
- Training in immunohistochemistry
- List of antibodies that are routinely used in the lab
Enzyme histochemistry--frozen muscles
- Cytochrome-C oxidase and SDH
- ATPase pH 9.6, NADPH, Alpha GDP
Embryology
- Expertise in mouse embryology
- Serial sectioning to structures
Procedures
Procedures and Optimal Fixation
Ideally, animals should be euthanized and tissues placed immediately into appropriate fixative
If you cannot necropsy the animal immediately:
- At the very least, put animal at 4C (DO NOT FREEZE)
- Preferably place entire animal in fixative (flat bottomed container)
- Open abdominal & thoracic viscera and snip skull
- fix (± gently inflating lungs and GI with fix)
- Make incisions in kidneys and liver if possible
Fixation methods
- Perfusion fixation followed by immersion fixation
- Immersion fixation (do not re-use fixative)
- Ideal fixative-tissue ratio 10:1—thus the mouse in a 50 mL conical is not ideal (use a flat bottomed container)
- Tissues should be no thicker than 3 mm for optimal fixation
- Bones should be cracked to allow for fixation of marrow
- Infusion of nasal cavities for optimum fixation of nasal structures
- Inflate lungs and intestinal tract with fixative
- If eyes are primary interest, need to identify appropriate fixative for your interests
- If testes are primary interest, consider Bouin’s
Fixative options
10% Neutral Buffered Formalin
Zinc Formalin
4% Paraformaldehyde
Bouin's Solution
Frozen tissues (Frozens)
Fixation Options
10% Neutral Buffered Formalin
- Routine fixative (Supplied free in our facility!)
- Don’t use 10% formalin (unbuffered)—this will destroy your data
- Done at room temperature
- Adequate for many antibodies (know before you fix!)
- Not adequate for most lymphocyte markers
- Fix no less than 24 hours but best if at least 72 hrs or longer; transfer to 70% EtOH to bring to lab
- Best to embed as soon as possible, but can stay in EtOH l
4% Paraformaldehyde (a buffered solution!)
- Commonly used in perfusion protocols
- Good for in situ hybridization (formalin works well, too)
- More gentle and considered slightly better than formalin for IHC, FISH
- Must be made just prior to use
- Keep it a 4C
Zinc Formalin (a buffered solution!)
- Commonly used for IHC when frozen not a good option
- More gentle and considered slightly better than 10% neutral buffered formalin for IHC
- Fix at room temperature
Bouin’s Solution (yellow)
- Contains picric acid l
- Maintains good morphology (good for brain)
- Decalcifies bone while fixing tissue
- May result in slight hardening of tissue if over-exposed (get into 70% EtOH within 48 hours)
- Colors tissue and affects some histochemical stains
Frozen tissues
- Freeze fresh or fixed tissues
- Good for immunohistochemistry for problem antibodies
- Decreased quality of morphology, better if pre-fixed
- Not good for bone (can’t cut the tissue)
- Store at -80
Paraffin Wax Blocks
- Rapid and correct fixation of small pieces of tissue are the key to well preserved morphology.
Tissue processing through graded alcohols and xylene are automated in
our Leica Tissue Processor, while paraffin embedding in the orientation
of the principle investigator’s choice is done by hand at the embedding
center.
- Sections of the resulting wax blocks can then be cut at 2-10µm to
provide single cell layer sections that are ready for chemical stains,
immunohistochemistry, ISH, as well as PCR and laser capture.
Frozen Block
Frozen section histology requires the use of fixed or un-fixed tissue samples
- If tissue is not being preserved by a fixative, it is imperative that the tissue is frozen as quickly as possible after removal from the animal
- Freeze in isopentane chilled to at least -80 in dry ice
Tissue samples should be small (3mm x 3mm) to avoid slow freezing of the sample, which can result in artifacts
- Dab tissue on paper towel to dry to reduce crystal formation during freezing
- Place tissue into mold and surround with OCT
- Remove bubbles
- Place mold in isopentane with flat face down, when frozen, drop mold into chilled isopentane
- If not pre-fixed, fix sections in cold 100% methanol or acetone for 2-3 minutes and store at –80 or –20C
Frozen tissues with special needs
- Frozen brains—how to get rid of bubble artifact
- Best to fix brains by immersion or perfusion followed by immersion lPlace brains in a 30% sucrose solution in PBS O/N (no longer) lEmbed in OCT and freeze as described
- Frozen muscle
- Prepare 2-methylbutane chilled in liquid nitrogen (2-methylbutane must be very cold such that it is slightly frozen on the bottom of the container before you freeze the muscle)
- Don’t pull or break muscle during dissection
- Using a small drop of OCT, adhere one end of the muscle to cork or cardboard
- Turn upside down and immerse in 2-methylbutane for 15-25 seconds and then place on dry ice (if you leave it too long in this super-chilled reagent, the sample will crack)
- Store at -80
- NEVER cover it with OCT! These must be frozen very quickly to avoid severe artifacts. Muscles can be frozen in liquid nitrogen as well