The histopathology section of the Animal Health Centre is responsible for the routine preparation of stained tissue sections mounted on glass microscope slides.
Many tissues are derived from necropsies completed within the Animal Health Centre, while others originate from fixed tissues submitted by veterinary practitioners throughout British Columbia. Prepared tissue sections encompass all varieties of animal species, including fetal tissues.
Tissues are trimmed from necropsy specimens previously fixed in 10% neutral buffered formalin. Overnight, automated tissue processors take the tissues through increasing concentrations of ethanols followed by xylene and, finally, into molten paraffin wax. Tissues are then embedded into molds and cooled in the freezer. The resulting blocks are sectioned at 3-6um thick using a manual microtome and mounted onto glass microscope slides. After spending 35 minutes in a 65°C oven, they are placed onto the automated stainer.
Sections are stained routinely with hemotoxylin and eosin (H&E) prior to microscope examination. Stained tissue sections are ready for examination by the pathologist approximately twenty-four hours after fixed tissues are forwarded to the Histopathology section. Specific diagnostic tests using special stains may also be used, if required. Immuno-histochemistry staining for specific pathogens (disease-causing agents) has been introduced to assist the pathologist by directly identifying these pathogens in tissue sections.
Histopathology - Submission Requirements
If histopathology is required, please try to fix samples in formalin at the time they are taken. Shipping samples fresh will result in rotting during transit, which will impeded microscopic tissue examination. Please also avoid freezing samples, as freezing damages the tissue and will also impede microscopic examination.
Preparation of fixed tissue with formalin:
Specimens should be no thicker than 5 mm at the thickest point. Other dimensions (e.g. length) are not critical; however the sample should be large enough to provide an adequate field of study. For larger tissues that must be submitted in-tact (e.g. brain), it is best to make several deep cuts into the tissue so that the formalin can penetrate more quickly.
A 10 to 1 ratio of formalin to tissue (by volume) is essential for adequate fixation. Samples should be allowed to fix in 10% neutral buffered formalin for at least 24 hours.
Once the sample is fixed, it can be transported using just enough formalin to cover the tissue. Alternatively, this tissue can be wrapped in formalin-soaked paper towel.
Recipe for 10% Neutral Buffered Formalin
- Formaldehyde 35-40% strength - 10 ml
- (Na H2 PO4 H2O) Sodium phosphate monobasic monohydrate - 0.4 gm
- (Na2 H PO4) Sodium phosphate dibasic anhydrous - 0.65 gm
- Water - to 100 ml