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Histology Syed > Tissue Prep and Staining > Flashcards

Tissue Prep and Staining Flashcards

1
Q

What are the major steps that need to be taken for tissue fixing and embedding?

A
  • Fixing - Dehydration - Removal of alcohol - Embedding
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2
Q

Tissue Fixing Step:

A
  • Fixing prevents further deterioration of the tissue specimen and helps to harden the tissue prior to embedding and sectioning - Give greater optical contrast - Formalin is one of the most widely used fixing agents
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3
Q

Whys is formalin a good fixing agent?

A
  • It may be used alone or in combo with other agents - alcohol (shrinks tissue) and/or acetic acid (softens and counteracts alcohol) - It reacts with amino acids of tissue protein and stabilizes tissue structure to prevent deterioration
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4
Q

What is something that formalin might not be used for?

A

Formalin is not good if fine cytological detail is desired

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5
Q

What do acid fixatives do and what are some examples?

A

They fix chromatic, nucleoli, and spindle fibers, but not mitochondria or nucleoplasm Ex: Carnoy’s fluid, Zenkers’ fluid, Bouin’s fluid

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6
Q

Acid Fixatives: Carnoy’s Fluid

A
  • mixture of chloroform, alcohol, acetic acid. It’s a good general fixative used for preserving glycogen in animal tissues
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7
Q

Acid Fixatives: Zenker’s Fluid

A
  • mixture of potassium dichromate, acetic acid, mercuric chloride. Gives great details. Have to be careful with washing because otherwise black crystals will form.
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8
Q

Acid Fixatives: Bouin’s fluid:

A
  • has picric aid, formalin, and acetic acid. Its a good general fixative that gives good cytological detail. Requires a prolonged and careful washing cycles **Picric acid can make your tissues yellow. It is also highly explosive in crystal form
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9
Q

What do basic fixatives do and what are some examples?

A

Basic fixatives can be used to fix tissues where mitochondrial staining id desired. In this procedure, chromatin is dissolved Ex: Zirkle-Erliki Fixative -

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10
Q

Basic Fixatives: Zirkle-Erliki Fixative

A

Contains potassium dichromate, ammonium dichromate, copper sulfate, and distilled water - requires a long fixing time (2 days)

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11
Q

What are some fixatives for TEMs?

A

Glutaraldehyde: Preserves proteins by cross-linking them Osmium tetroxide: Reacts with lipids (phospholipids) and imparts electron density to cell and tissue structures

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12
Q

Step 2: Dehydrating Why do we dehydrate?

A

Because the tissue sample will eventually be embedded and infiltrated with a hydrophobic material (usually paraffin), all the water must be removed from the tissue

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13
Q

How do we dehydrate tissues? What are some examples of agents?

A

We place the tissue in successively increasing strengths of ethanol until water is gone. **Ethanol dissolves neutral fats, so it can’t be used for dehydration if we want those** Ex: N-butyl alcohol or acetone

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14
Q

Step 3 of Fixing: Clearing - Why/How do we clear?

A

We are replacing the alcohol with an agent such as xylene or cedar oil Note: The paraffin embedding medium will not mix with alcohol but will mix with clearing agents

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15
Q

Step 4: Embedding What do we do?

A

The tissue is moved sequentially through several (3) melted paraffin baths - Afterwards, it is placed in a mold that is then filled with melted paraffin - the paraffin mold is hardened by pacing it in a cold water bath (have to do it just right, not too early or late)

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16
Q

How do we embed for TEMs?

A
  • Tissues are infiltrated with a monomeric resin (epoxy resin) - Resin is then polymerized - Tissue samples are typically less than 1mm3
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17
Q

How is tissue sectioning done?

A

We move the paraffin block up and down. It is clamped down into place. Each time we move it up and down, you cut a section of the tissue. The next section you make, it has just enough friction to create heat and stick to the first section. So, you can put up to 4 or more sections on the slide together for more efficient viewing

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18
Q

How is tissue sectioning done for TEMOL?

A
  • Sections are cut using diamond knives at 50-150nm - Sections too fragile to handle - must be floated onto a plastic-coated copper mesh grid - Holes in copper grid allow the electron beam to pass through
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19
Q

Give a general tissue staining prep outline:

A
  • paraffin must be removed (done through xylene) - xylene must be removed using a graded series of alcohol down to water - Stains are applied and is dehydrated again through alcohols - Alcohol is removed with xylene - Drop of cement followed by cover slip
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20
Q

What does the stain H&E stand for?

A

Hematoxylin(behaves like a base) and eosin (acid dye)

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21
Q

Explain some of the characteristics of H&E stains:

A

H&E are commonly used for routine staining because they show structural features well, but do NOT say much about the chemical characteristics of the tissues - hematoxylin behaves like a base because of the properties of the mordant that is used to help it bind to the tissues - Eosin an acid due, stains most the cytoplasmic components and much of the extracellular material a yellowish pink

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22
Q

Why are we not worried about H&E stains not telling us much about the chemical characteristics of the tissues?

A

Because there are hundreds of other stains that will tell us things about the chemical characteristics

23
Q

What are the dark circle and why do they show up?

A

ADD PIC The dark circles are nuclei. They show up because of the H&E.

24
Q

Examples of stains: What are orcein and resorcin fuchsin stains used for?

A

Orcein and resorcin fuchsin stains can be used to reveal elastic material

25
Q

Examples of stains: What is silver impregnation used for?

A

silver impregnation is used to show reticular fibers and basement membranes

26
Q

What is an example of a fat soluble stain for lipids?

A

Sudans

27
Q

Explain how basic dyes work

A

Basic dyes react with the anionic groups of tissue components such as phosphate, sulfate, and carboxyl groups. Can bind to RNA???? - Exact nature of binding depends on the pH** At a high pH, all 2 groups are averrable for binding with the dye - Any tissue that reacts with a basic dye is called basophilic

28
Q

What are some examples of basic dyes?

A
  • Methyl green, methyl blue, pyronine G, toluidine blue
29
Q

What effect can pH have on staining?

A

It can change the dye color (add pic slide 20)

30
Q

Explain how acidic dyes work

A

Acidic dues bind to tissue components by forming electrostatic linkages with cationic groups such as the amino groups of proteins.

  • Different types of acid dyes have slightly different properties and can be used in sequence to give different results. (So these show the protien components of cells)
31
Q

What is Mallory’s triple stain dye?

A

Three acid dyes

  • Aniline blue - stain collagen
  • acid fuchsin - stains ordinary cytoplasm
  • Organge G - stains red blood cells
32
Q

What do you call any tissue component that reacts with an acid dye?

A

Acidophilic

33
Q

What are some examples of acid dyes?

A
  • Acid fuchsin
  • Aniline blue
  • Eosin
  • Orange G
34
Q

What does Metachromasia mean and what is an example?

A

It refers to when a dye changes color after reacting with a tissue component.

Ex: Toluidine Blue - used to stain cartilage ground substance or mast cell ganules, changes to a different shade of blue or a violet

35
Q

Staining for TEM viewing used ions of heavy metals that are very electron-dense. When can these heavy metals be added and what is an example?

A

Heavy metals can be added during fixation, dehydration, or by doacking in ionic solutions after sectioning

Ex: Ocmium tetroxide

36
Q

What is the Perls’ Reaction?

A

The Perl’s reaction is used to demonstrate the presense of iron in tissues. Especially in patients with diseases that store iron (ex: hemochromatosis)

  • It incubates tissues in a mixture of potassium ferrocyanide and HCl. THe results are an insoluble blue presipitate of ferric ferroxyanide
37
Q

How and why do stains for lipids have to be done differently?

A

Lipids are soluble in the reagents that are normally used in the processing of tissues for histological examination

  • So instead, we use frozen sections to geive the best lipd viewing results.

Ex dyes for lipids: Sudans, oil red O, and nile blue

38
Q

Give a clinical examply of how lipid staining may be used during surgery

A

During surgery on a tumor area, they’ll take a frozen section to the lab to find out if it is malignant or not. If it is/isn’t, they would adjust the procedure

39
Q

What is this a stained example of?

A

Adipose (osmium tetroxide)

Slide 27

40
Q

Histochemical Staiing Techniques:

Schiff reagent reactions:

Feulgen reaction:

A
  • The schiff reagent reaction depends on the formation of aldehyde groups following exposure to HCl or periodic acid.

Fuelgen reaction: Mild hydrolysis with HCl exposes aldehyde groups on deoxyribose

  • Schiff reagent reacts with the aldehyde groups and forms a deep-pinkish color
41
Q

What stain and organ is this an example of?

A

This is the liver and the nuclei have been dyed with the schiff reagents

  • Ask him about this slide

Slide 30 - ADD PIC

42
Q

Histochemical Staining Techniques:

Schiff reagent reactions:

Period acid-Schiff reaction (PAS):

A
  • Periodic acid is used to cleave bonds between adjacent carbons of carbohydrates and form aldehyde groups
  • Schiff reagent reacts with the aldehyde groups and forms a deep-pinkish color
43
Q

What are some examples of Periodic acid-Schiff reaction (PAS) substances?

A
  • Polysaccharides (glycogen)
  • Glycosaminoglycans
  • Proteoglycans
  • Glycoproteins
  • Glycolipids
44
Q

What is a clinical application of the Periodic acid-Schiff reaction (PAS)?

A

Helpful in biopsies of tissues from patients with glycogenoses (glycogen storage diseases)

  • ADD PIC SLIDE 32
45
Q

What is Best Carmine?

A

Best carmine is a dye that may also be used to demonstrate glycogen deposits

ADD PIC Slide 33

46
Q

How are RNA-stains different from other stains?

A
  • RNA-rich organells may be stained with basic dyes
  • For these, you need control slides to distinguish other basophilic substances
  • Control slides are incubated with rinonuclease
47
Q

How are Immunocytochemical Staining Techniques different from others?

A
  • Immunocytochemical techniques can be used to study the presence of specific tissue constituents (antigens) by using monoclonal antibodies
48
Q

What are monoclonal antibodies?

A

They are derived from activated B cell clones exposed to a specific anigen. These antibodies are very specific.

  • Most antigens have a variety of epitopes (binding sites) that generate a number of different antibodies - polyclonal
  • A single immune response to an antigen is referred to as monoclonal
49
Q

What are examples of antigens for immunocytochemical techniques?

A
  • Protiens, glycoproteins, proteoglycans
50
Q

How do we produce a hybridoma?

A

Well, B lymphocytes can mtate into tumor cells resulting in a myeloma.

  • Myeloma cells acquire the ability to grow indefinitely in culture
  • My fusing a single activated B cell and amyeloma cell - you create a hybridoma that can grow indefnitely in culture AND produce a speciic monoclonal antibody.

Best of both worlds

51
Q

What is direct labeling for antibodies?

A

Antibodies can be conjugated with various things to produce a visible marker in various microscopes

Ex:

They can be conjugated with a floursescent dye to produce a visible marker in flourescent microscopy

They can be conjugated with a visible substance to produce a visible marker in light microscopy

They can be conjugated with gold or ferritin to produce a visible marker in electron microscopy

52
Q

What is indirect labeling for antibodies?

A

In this variation, the marker is attached to a second antibody which is specific to the antibody used to locate the antigen of interest

53
Q

What does it mean when a cell is stained a certain color while stained with (for example: anti-insulin)

A

It means that the cells that show up dyed, are the ones that produce insulin. This technique can be used for the pancreas (anti-insulin and anti-glucagon), Pituitary (Anti-ACTH), and Tonsils (Anti-IgG)

ADD PICS - slides 41-43

Histology Syed (6 decks)

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