Histology Wk 1 Flashcards
What is histology
The study of tissues of the body
What are the two interacting components of tissues
Cells and extra cellular matrix (ecm)
What does the ecm consist of…
Many kind of macromolecules which form complex structures such as collagen fibrils
It supports cells
Name the steps for preparation of tissue for study
Fixation
Dehydration
Clearing
infiltration
Embedding
Trimming
What is fixation
Small pieces of tissue are placed in solutions of chemicals that cross link proteins and inactivate degradative enzymes, which preserve the tissue structure.
Because fixative must fully diffuse through the cell it is cut into small fragments before fixation to facilitate penetration
What is dehydration
The tissue is transferred through a series of increasingly concentrated alcohol solutions ending in 100 percent which removes all water
What is clearing
Alcohol is removed in organic solvents in which both alcohol and paraffin are miscible
What is infiltration
The tissue is then placed into melted paraffin in an oven at 52-60 degrees until it becomes completely infiltrated with this substance
What is embedding
The paraffin infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden
Tissues are embedded with plastic resin are also dehydrates in ethanol and then dehydrated in ethanol and infiltrated with plastic solvents that harden when cross linking polymerisers are added
What is trimming
The resulting paraffin block is trimmed to expose the tissue for sectioning on a microtome
What is a microtome
It is used for sectioning paraffin embedded tissues for light microscopy
What are paraffin section usually cut into
3-10 micrometers thickness for light microscopy and less than 1micrometer for electron microscopy
What is the term basophilic
Cell components such as nucleus acids with negative negative charge have an affinity for basic dyes
What is the term acidophillic
Cationic components such as proteins with many ionized amino groups stain more readily with acidic dyes
Name some acidophilic and basophilic components
Basophilic
DNA
Rna
Glycoaminoglycans
Acidophilic
Mitochondria
Secretory granules
Collagen
What is hematoxylin used for
Stains dna in the cell nucleus, rna rich portions of the cytoplasm and the matrix of the cartilage producing a dark blue or purple color
What is eosin used for
Stains cytoplasmic structures and collagen pink
Describe the period acid- Schaffer reaction (PAS)
Utilises the hexose rings of polysaccharides and other carbohydrate rich tissue structures and stains such macromolecules purple or magenta
What is autoradiography
It is a method of localizing newly synthesized macromolecules in cells or tissue sections
What are radioactively labeled metabolites
Nucleotides
Amino acids
Sugars
How are slides with radioactively labeled metabolites produced
Slides containing these are coated in a darkroom with photographic emulsion in which silver bromide crystals act as micro detectors of the radiation
Silver bromide crystals are reduced by radiation to produce small black grains of metallic silver either under LM OR TEM which indicate the locate the radio labelled macromolecules in the tissue
What is in vitro in cell and tissue culture
Live cells can be maintained and studied outside the body in culture
What is in vivo in cell and tissue culture
Cells are bathed in fluid derived from blood plasma and containing many different molecules required for survival and growth.
What is enzyme histochemistry
Is a method for localizing cellular structures using specific enzymatic activity present in those structures
What are the steps for enzyme histochemistry
- Tissue sections are immersed in a solution containing the substrate of the enzyme to be localized
- The enzyme is allowed to act on its substrate
- The section is then put on contact with a marker compound that reacts with a product of the enzymatic action on the substrate
- The insoluble structure is visible by light or electron miscroscopy
Visualizing specific molecules
Labels are fluorescent compounds, radioactive atoms that can be detected with autoradiography
Molecules of peroxidase or other enzymes can be detected with histochemistry
Metal particles can be seen with light and electron microscopy
What is immunohistochemsitry
A highly specific interaction between macromolecules is that between an antigen and its antibody
To localize and identify many specific proteins
What is an advantage of using monoclonal antibodies rather than polyclonal antibodies
It can be selected to be highly specific and bind strongly to the protein to be detected
What is the method of immunohistochemistry
A tissue section that one believes contains the protein of interest is incubated in a solution containing antibody against the protein
The antibody binds specifically to the protein and after a rinse the protein’s location in the tissue can be seen with a light of electron microscope
LOOK AT NOTEBOOK IN GOODNOTES
What are the two methods of immunocytochemistry
Direct and indirect
What is the direct method
If the cell or tissue antigen of interest is detected by directly binding a labeled primary antibody specific for that antigen
Fluorescent antibodies bind to the direct target antibody
What is the indirect method
Uses an unlabelled primary antibody that is detected bound to its antigen with labeled secondary antibodies
Why is the indirect method used more widely in research and pathologic tests
It is more sensitive with the extra level of antibody binding serving to amplify the visible signal and cheaper
What is hybridisation
The specific binding between two single strands of nucleus acid, which occurs under appropriate conditions if the strands are complementary
What is in situ hybridisation
When nucleic acid sequences in solution are applied directly to prepared cells and tissue sections
What is in situ hybridisation ideal for
Determining if a cell has a specific sequence of dna such as a gene
Identifying the cells containing specific mRNA
Determining the localisation of a gene in a specific chromosome
Probes
The nucleotide sequence of interest is detected with probes
The probe is obtained by cloning by polymerase chain reaction
Why is Sudan black used
Lipid soluble
What fixative is used in light microscopy
37% formaldehyde
What fixative is used in EM
Glutaraldehyde reacts with the amine groups of proteins
