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Histology: branch of biology which studies microscopic anatomy of biological tissues

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Fluorescence Microscopy:
- Use of fluorescent dyes which absorb light and project it at a different wavelength to detect certain proteins/structure in tissues
- Used fluorescent microscope

Filters:

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- Biopsy: remove tissue from where you want to analyze
- Slicing: once tissue is remove slice into small sections
- Fixative: place fixative onto tissue so they will bind to animo acid groups on proteins so tissue doesn't degrade
- Freeze: freeze is resin
- Slicing: slice smaller sections to view in electromicroscope

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Anaplastic Cells:
- used to describe cancer cells that divide rapidly and no longer resemble normal cells
- If ratio to nucleus and cytoplasm ratio is 1:1 because increases mitotic activity

Hyperplasia:
- Increase number of cells but cells are still relatively/appear normal

Adenoma:
- When there is an epithelial tumor with glandular organisation.
- Glandular tissue: anything that is hollow/glands
- Benign tumors

Early Carcinoma:
- Carcinoma: most common type of malignant cancer coming from epithelial cells
- Can also be called insito --> not

Late Carcinoma:
- Later stage carcinoma
- Can be called invasive --> expanded and invaded surrounding regions
- Can go into lymph nodes and blood stream, etc

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- Epithelial Cells: cell that lines a surface of your body. Found on skin, organs, etc.
- Functions:

- Basement Membrane: Thin sheet which is type of extracellular matrix which provides cell and tissue support
- Act as place for complex signalling
- Between epithelial cells and connective tissue
- Has certain chemical structure: mostly glycoproteins, collagen
- Is made up of 2 layers: basal lamina and reticular lamina

- Myoepithelium: Typically found in glandal epithelium above basementment membrane. Cells that can be found in salivary glands, sweat glands, mamory glands, etc.
-Smooth muscle properties
-Helps for lactation

- Intralobular stroma: Contains fibroblasts and scattered inflammatory cells

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Benign tumor: stays inside basal lamina
Malignant tumor: breaks basal lamina and becomes invasive
Basal lamina refers to a layer of basement membrane (not cells just layer of fibers)

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Biomarker:
- Molecule found in blood/any bodily fluid that shows a normal or abnormal process to help measure/observe progress of a disease

Can organelles be used as biomarkers?:
- Yes because any biological molecule indicating abnormality
- E.g. if a nucleus is abnormally large that gives indication that something is wrong

In immunohistochemistry:
- The biomarkers are used to show how many/where of a receptor there are

Histologic:
- Abnormal alterations in cells and tissues
- Used in grading stages of cancer

Molecular:
- Biophysical properties
- E.g. blood glucose

Radiographic:
- Found through imaging studies
- Can use lead wire because it lights up in radiograph so you can see exactly what you marked
- E.g. bone mineral density

Physiologic:
- Certain physiological process
- E.g.l blood pressure

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H&E Staining:
- Uses 2 dyes

Hematoxylin:
- Basic dye which binds to acidic structure with purple/blue structure
- DNA, RNA, ribosomes

Eosin:
- Acidic dye used to dye basic structures red/pink
- Mainly binds to proteins within cytoplasm

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Steps:
1) Remove wax:
- After preparing the slides, elements are infiltrated with and surrounded by paffix wax that is hydrophobic
-Wax removed by hydrocarbon solvent (xylene)

2) Hydrate section
- Tissue slide is passed through alcohol to remove xylene solvent and rinsed in water

3) Apply hematoxylin nuclear stain:
- Slide stained with hematoxylin (contains dye of hermatein and binding agent)
- This will stain nuclei and other nuclear elements a purple color

4) Complete nuclear staining by "bluing":
-Sample is rinsed and 'blued' by weakly alkaline solution
-This will make hematoxylin a dark blue

5) Remove excess background stains
- Removal of nonspecific stains
- Help make specific stains clearer and increases contrast between
- Done with weak alcohol with water again

6) Apply eosin counterstain:
- Stain section with eosin
- Will make nonnuclear elements pink

7) Rinse, dehydrate, clear, & mount:
-Samples passed through alcohol then rinsed with xylene to make tissue transparent
-Thin layer of polystyrene mountain applied

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Immunohistochemistry:
- Use of antibodies to visual tissues and cells and their functioning
- Put markers on antibodies to signal where a protein/structure is on cell

ER & PR:
- Negative: no biomarker or antibody
- Positive: there is biomarker or antibody
- ER: estrogen receptor // PR: progesterone receptor
- ER and PR are biomarkers
- ER positive they require estrogen for cancer cells to keep growing
- PR positive means they require progesterone for cancer cells to keep growing

- Patient with malignant breast tumor (either ER or PR):
- What does this mean reegarding treatment?
- Start with control of hormonal releases (blocking) to prevent tumor growing

Relating to breast cancer:
- Can see if there is a specific antigen expressed in tissues

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Steps:

1) Tissue is fixed with certain chemical (formaldehyde) which ensures structural integrity of the cell

2) Cells are penetrated with Triton X allowing antibodies to bind with tissues

3) Primary antibodies against desired protein are added and secondary proteins with enzymes (like HRP) bind to the primary antibody
- The enzyme is conjugated so it is attached

4) Secondary enzymes target certain substrate and catalyze oxidation which results in colorful staining of the area

5) Counterstain added to tissue (like hematoxylin) which makes contrast with what was stained in immunohistochemitry steps with what was not stained

ER/PR:
- We have a breast biopsy,
- First antibody binds to ER or PR receptor (that is the antigen here)

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Figure A: electrons when they absorb energy they jump. When they fall back to original energy state they emit light

Figure B: After all light sources go in , the first barrier only allows certain wavelength of light to go through (450-490nm). Then it is deflected by beam-splitting mirror. Will excite the sample with fluorescent probes. When object is excited the emitted light will go to second barrier filter which will cut out unwanted fluorescent signals which go to microscope eyepiece to be seen (these are between 520-560nm).

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What would be seen in H&E staining of anaplastic cells

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What would be seen in H&E staining of anaplastic cells

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-Larger nucleus
- Cytoplasm to nucleus ratio: less cytoplasm more nucleus
-Hyperchromatic nuclei: Nucleoli would be more prominent
- Morphism: normal cells have shape related/similar to one another vs anaplastic cells have morphism (sizes of nucleus and cell shape are very irregular)
- Abnormal tissue architecture: E.g. blocking the lumen
- Giant cells: When cells are very close to each other (because of rapid expansion) they can form giant cells by combining. So you have 1 giant cell with multiple nuclei