Full Procedure for H and E Staining
Paraffin processing of tissue
Fixation of Tissues
1. Where the best possible morphology is required, animals should be anesthesized and subjected to cardiac perfusion with saline, followed by a 10% formalin flush. If biochemical studies need to be performed on the tissue, a 10% formalin flush should not be used as it may interfere with subsequent analysis.
2. For routine stains where perfusion is not required, tissue is sectioned and drop-fixed in a 10% formalin solution. Fixative volume should be 20 times that of tissue on a weight per volume; use 2 ml of formalin per 100 mg of tissue.
3. Due to the slow rate of diffusion of formalin (0.5 mm hr), tissue should be sectioned into 3 mm slices on cooled brain before transfer into formalin. This will ensure the best possible preservation of tissue and offers rapid uniform penetration and fixation of tissue within 3 hours.
4. Tissue should be fixed for a minimum 48 hours at room temperature.
5. After 48 hours of fixation, move tissue into 70% ethanol for long term storage.
6. Keep fixation conditions standard for a particular study in order to minimize variability. (Although set times are best, tissue may be fixed for substantially longer periods without apparent harm.