Histological Staining and its Methods

Perspective - Journal of Interdisciplinary Histopathology (2022)

Histological Staining and its Methods

Daniel Chen*
Department of Pathology, Public University in Valencia, Valencia, Japan
*Corresponding Author:

Daniel Chen, Department of Pathology, Public University in Valencia, Valencia, Japan, Email:

Received: 03-Sep-2022, Manuscript No. EJMJIH-22-73902; Editor assigned: 05-Sep-2022, Pre QC No. EJMJIH-22-73902 (PQ); Reviewed: 19-Sep-2022, QC No. EJMJIH-22-73902; Revised: 27-Sep-2022, Manuscript No. EJMJIH-22-73902 (R); Published: 05-Oct-2022


In pathological diagnosis and forensic investigations, histological staining is a frequently used medical procedure. Fixation, processing, embedding, sectioning, and staining is the five main steps in the histological staining procedure.

The microscopic study of animal and plant cells and tissues using staining, sectioning, and microscopy is known as histology (electron or light microscope). The properties of the tissue and the microscopic cell structures are studied using a variety of techniques. Forensic investigations, autopsies, diagnostics, and education all involve histological studies. Histology is also widely utilized in medicine, particularly when examining sick tissues to help with treatment.

Histological staining is a set of procedures used to prepare sample tissues for microscopic examination by staining them with histological stains. Fixation, processing, embedding, sectioning, and staining is the five main steps in the histological staining procedure. By combining chemical, molecular biology assays, and immunological procedures, significant advances have been made to the methods used for histological staining, which have substantially aided in the study of organs and tissues.

Specific histopathology features

Staining: Staining is used to draw attention to significant tissue characteristics and to improve tissue contrast. There are, however, a number of different staining techniques that are utilized for specific cells and components. A typical medical procedure for locating sick, tumorous, or other pathological cells is staining, which involves applying a dye color to the rear and anterior boundary of sample tissues. Staining is used in biological research to identify cells, nucleic acids, proteins, and gel electrophoresis to facilitate microscopic study. Differential staining, double staining, and other multiple staining techniques may be employed under certain circumstances.

Fixation: Fixation in histology refers to the application of chemicals to protect the cellular structure from deterioration and preserve the organic tissue structure. When doing the investigation under a light microscope, neutral buffered formalin is typically used. Fixatives improve tissue and cell preservation by cross-linking proteins in an irreversible manner. The fixation phase preserves the chemical makeup of the tissues, makes cells or tissues more rigid for cutting, and postpones deterioration. Fixatives also alter tissue penetration and affect antigen exposures, which can be advantageous or harmful. Two methods are used to give these fixatives: perfusion and immersion of the prepared tissue. Through diffusion, these fixatives are absorbed by the animals’ bodies. One fixative can only be used at a time during perfusion, which takes more time. Although there are other fixatives in use, formaldehyde fixatives are the most popular.

The Neutral Buffered Formalin (NBF) provides good tissue and cell structure preservation while stabilizing amino acids in proteins. Although useful for immunostaining, paraffin-formalin (paraformaldehyde-PFA) must be freshly produced to maximize its potency. It has been discovered that the Bouin fixative works well on delicate and sensitive tissues, including tiny tissues, embryonic tissues, and brain tissues. Although Bouin fixative penetrates slowly and distorts kidney tissues and mitochondria, it gives good preservation of nuclei and glycogen.

Dehydration: The goal of this phase is to remove water from the chosen tissues in order to harden them and make it easier to cut them into thin sections for use in light and thick sections for use in electron microscopes. The dehydration process uses ethanol to draw water out of the tissues. To get rid of the alcohol, paraffin wax, and contaminating agent, the procedure is repeated through a hydrophobic clearing agent such xylene. Resins are utilized to improve the cutting of delicate tissue sections.

Embedding: This is accomplished using paraffin wax to make it simpler to extract cellular components. Plastic resin, wax, or combinations of fixatives are used to achieve good morphology in complicated biological tissues. However, the prolonged heating caused by these fixatives may result in the breakdown of the cell and tissue structures, which could cause issues with the hybridization process because of the unstable RNA. In a similar vein, the penetration of paraffin wax results in the suppression of antibody, chemical, and other fixative penetration. After embedding tissues, freezing them, removing the wax after staining, and using PFA fixatives are effective ways to solve this issue and achieve enhanced morphology.

Sectioning: In histology, sectioning is the process of cutting a tissue into ‘ribbon-like’ microtomes in order to put it on a microscope slide for examination. In this instance, a number of thin tissues sections are cut and processed using the paraffin procedure. Because the necessary chemicals have been shown by science to be harmful, some staining techniques have been abandoned. Similar variations in workload necessitate the use of increasingly sophisticated staining techniques. The case studies show that a mix of various stain procedures is utilized in contemporary histology to increase the staining process’ efficacy. Several stains have been modified and coupled with other stains in the modern histology to increase the effectiveness of histological stains.

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