PS1_Histo_Lect4_Part 1_Power Point Presentation.pptx

PATHOLOGICAL SCIENCE 1 Staining Principles Lecture 4 Miss Yulia Humrye, BSc (Hon) [email protected] CONTENTS  Introduction to staining principles  Theory of histological staining  Staining – main types of action Name of the book? Author? I showed my masterpiece to the grown−ups, and...

PATHOLOGICAL SCIENCE 1 Staining Principles Lecture 4 Miss Yulia Humrye, BSc (Hon) [email protected] CONTENTS  Introduction to staining principles  Theory of histological staining  Staining – main types of action Name of the book? Author? I showed my masterpiece to the grown−ups, and asked them whether the drawing frightened them. But they answered: "Frighten? Why should any one be frightened by a hat?" ART OF PATHOLOGY COMPET ITION 'UNITY' BY MEREENA PUN (UK) 'AGAINST IT ALL' BY WERONIKA KUKULSKA (UK) 'PILLARS' BY DR EMMA ALLDE (UK) WHY DO WE NEED TO STAIN TISSUE SECTIONS?  When tissue sections have been prepared they are almost transparent.  To obtain useful clinical information tissue sections need to be stained and various tissue elements need to be selectively demonstrated.  There are many methods for selectively demonstrating tissue elements within tissue sections, that can be divided as: 1. Routine morphological staining 2. Special Stains 3. Vital stains 4. Immunohistochemistry 5. In situ molecular methods Simple columnar epithelium Epithelium – colonocytes Intestinal gland Goblet cells No villi Mucosa Lamina propria Submucosa MALT Muscularis mucosa Adipocytes Duodenal Biopsy Duodenal Biopsy V LP C LP C Micrograph showing the villi (V) protruding into the small intestine lumen; note the crypts (tubular glands) (C) extend down from the base of the villi. The lamina propria (LM) is most clearly seen in the core of the villi, but also surrounds and supports the gland crypts. Gluten enteropathy (coeliac disease). Micrograph of villous damage resulting from gluten sensitivity. The mucosal surface is flattened owing to extensive loss of villi. The absorption function depends on the integrity of the villi. If enough villi are damaged, food material cannot be absorbed, leading to weight THEORY OF HISTOLOGICAL STAINING … NOMENCLATURE NOTE  Staining always involves the visual labelling of some biological entity by attaching, or depositing in its vicinity, a marker of characteristic colour or form.  The stain is the marker, or the reagent used to generate the marker. WHY AND HOW STAINING HAPPENS?  Stain uptake is often due to dye-tissue or reagent-tissue affinities: A tissue component has a high affinity for a dye merely means there is a tendency for a stain to transfer from solution onto a section.  Uptake of dyes and reagents is often multistep in both space and time.  A reagent may initially enter tissues due to coulombic attractions. Once inside it may form covalent bonds with some tissue grouping.  The intensity of staining may also be influenced by stain solubility in solvent and tissue environments.  Contributions to stain-tissue affinity are: Reagent-tissue interactions, Solvent-solvent interactions, Reagent-reagent interactions. The affinity of dyes for tissue elements is affected by a number of factors – The structure of the dye molecule – The shape of the dye molecule – The charge distribution of the dye – Solvent characteristics COMPOSITION OF TISSUES = CELLS Main component (80%) of cells is water Other components: Proteins (e.g. compose collagen, elastin, fibrin) DNA RNA Lipids Phospholipids Carbohydrates Glycoproteins Mucins Depositions (e.g. amyloid, calcium, iron) Pigments Infectious agents STAINING OF FORMALIN FIXED PARAFFIN EMBEDDED (FFPE) SECTIONS Wax must be removed first = sections to water (STW) Most staining solutions are aqueous = rehydrate There has to be a final dehydration step = dehydrate, clear, mount (DCM) Most stained preparations need to be permanent = coverslip using media (e.g. DPX) ONCE SECTIONS PREPARED THEY CAN BE REHYDRATED Xylene Xylene 100% Alcohol 90% Alcohol 70% Alcohol  The process is called taking sections to water.  This is the first part of many demonstration methods. Water ONCE SECTIONS STAINED THEY CAN BE DEHYDRATED Xylene Xylene 100% Alcohol 90% Alcohol 70% Alcohol  The process is called dehydration of sections.  This is the last part of many demonstration methods. Water