What is Neutral Buffered Formalin?
Neutral buffered formalin (NBF) is a widely used fixative in the field of histology. It is commonly a solution of 10% formaldehyde in a phosphate buffer, which helps maintain a neutral pH of 6.8 to 7.2. This buffering capability is crucial as it minimizes the formation of formic acid, which can cause tissue artifacts.
Fixation: It cross-links proteins, which stabilizes tissue structures and prevents degradation.
Preservation: It maintains the integrity of cellular components, making it easier to analyze tissue under a microscope.
Compatibility: It is widely compatible with various staining techniques and histochemical reactions.
How Does Neutral Buffered Formalin Work?
The main active component, formaldehyde, reacts with amino and other reactive groups in proteins to form methylene bridges. This cross-linking process stabilizes the tissue structure. The phosphate buffer ensures that the pH remains neutral, which is critical for effective fixation and minimizing tissue artifacts.
What are the Steps in Using Neutral Buffered Formalin?
1.
Specimen Collection: Tissue samples are collected and trimmed to appropriate sizes.
2.
Fixation: Samples are immersed in NBF, usually for 24-48 hours, depending on the size and type of tissue.
3.
Processing: Post-fixation, tissues are processed through a series of alcohols and clearing agents before embedding.
4.
Embedding: Fixed tissues are embedded in paraffin wax to facilitate sectioning.
5.
Sectioning: Thin sections of tissue are cut using a microtome and mounted on slides.
6.
Staining: Slides are stained to visualize different cellular components and structures under a microscope.
Toxicity: Formaldehyde is a toxic substance and requires careful handling and appropriate safety measures.
Over-fixation: Prolonged exposure can lead to over-hardening of tissues, which might make sectioning difficult.
Chemical Reactions: Formaldehyde can react with some tissue components, potentially interfering with certain staining techniques.
Alcohol-based fixatives: These are quicker but may cause tissue shrinkage.
Bouin’s solution: Effective for preserving delicate structures but contains picric acid, which is hazardous.
Glutaraldehyde: Offers better preservation of ultrastructure but is more expensive and less penetrative.
Conclusion
Neutral buffered formalin remains a cornerstone in histological procedures due to its effectiveness in preserving tissue morphology and cellular details. Despite its limitations, it is widely used because of its compatibility with various staining techniques and its ability to provide consistent results. Proper handling and adherence to protocols can mitigate its drawbacks, making it an invaluable tool in histology.
