Processing tissues for light microscopy 处理光镜用组织【University of Bristol】

• Fixation: Specimens may be routinely fixed in 10% neutral buffered formalin (NBF). Fixation should be commenced as soon after surgical excision as is practically possible to prevent autolysis and bacterial decomposition.

• Formalin fixation should ideally be allowed to proceed for at least 12 hours before processing. It is largely reversible by water washing until the specimens have been in formalin for more than 24-28 hours. Specimens may be stored in NBF.

• It is important to use NBF routinely as it stops the formation of formalin pigment in the tissue (caused by acidity in the fixative solution). This would otherwise present as artefact in the final stained sections.

• Very small specimens may alternatively be fixed in Bouin's fluid. Picric acid in the Bouin's fluid colours the tissue yellow temporarily, this facilitates subsequent handling before and after processing, and is easily removed from cut sections by alcohol in the initial steps of the staining procedure. It should be noted that tissues fixed in Bouin's tend to stain more brightly than those fixed in NBF.

• Decalcification: After fixation bone and other calcified tissues are treated with 10% formic acid to remove the calcium content. Tissues may be left standing in the acid for several weeks, changing the acid weekly until a chemical test for dissolved calcium is negative. Large specimens can then be further cut and trimmed to expose appropriate structures for subsequent processing.

• Phenol Solution: Other hard or dense material such as tendon, nail, decalcified bony structures etc., may benefit from pre-treatment with a mixture of 4% phenol in 70% alcohol (ie: 4g phenol dissolved into 100ml 70% alcohol), before final processing. This helps to soften the tissues for easier sectioning. It can also be used to 'rescue' tissue that has been accidentally dried out. The length of time the specimen is left in the solution depends on how badly dehydrated or hard the tissue is but generally 12-18 hours will be sufficient.

• Some softening of tissue and further surface decalcification may be performed on the wax embedded tissue if required. (Trimmed block surfaces are treated with a commercial solution of softening fluids known as Mollifex^® immediately before cutting.)