Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is something that formalin might not be used for?

A

Formalin is not good if fine cytological detail is desired

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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 -

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What does the stain H&E stand for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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
Examples of stains: What is silver impregnation used for?
silver impregnation is used to show reticular fibers and basement membranes
26
What is an example of a fat soluble stain for lipids?
Sudans
27
Explain how basic dyes work
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
What are some examples of basic dyes?
- Methyl green, methyl blue, pyronine G, toluidine blue
29
What effect can pH have on staining?
It can change the dye color (add pic slide 20)
30
Explain how acidic dyes work
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
What is Mallory's triple stain dye?
Three acid dyes - Aniline blue - stain collagen - acid fuchsin - stains ordinary cytoplasm - Organge G - stains red blood cells
32
What do you call any tissue component that reacts with an acid dye?
Acidophilic
33
What are some examples of acid dyes?
- Acid fuchsin - Aniline blue - Eosin - Orange G
34
What does Metachromasia mean and what is an example?
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
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?
Heavy metals can be added during fixation, dehydration, or by doacking in ionic solutions after sectioning Ex: Ocmium tetroxide
36
What is the Perls' Reaction?
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
How and why do stains for lipids have to be done differently?
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
Give a clinical examply of how lipid staining may be used during surgery
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
What is this a stained example of?
Adipose (osmium tetroxide) Slide 27
40
Histochemical Staiing Techniques: Schiff reagent reactions: Feulgen reaction:
- 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
What stain and organ is this an example of?
This is the liver and the nuclei have been dyed with the schiff reagents - Ask him about this slide Slide 30 - ADD PIC
42
Histochemical Staining Techniques: Schiff reagent reactions: Period acid-Schiff reaction (PAS):
- 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
What are some examples of Periodic acid-Schiff reaction (PAS) substances?
- Polysaccharides (glycogen) - Glycosaminoglycans - Proteoglycans - Glycoproteins - Glycolipids
44
What is a clinical application of the Periodic acid-Schiff reaction (PAS)?
Helpful in biopsies of tissues from patients with glycogenoses (glycogen storage diseases) - ADD PIC SLIDE 32
45
What is Best Carmine?
Best carmine is a dye that may also be used to demonstrate glycogen deposits ADD PIC Slide 33
46
How are RNA-stains different from other stains?
- 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
How are Immunocytochemical Staining Techniques different from others?
- Immunocytochemical techniques can be used to study the presence of specific tissue constituents (antigens) by using monoclonal antibodies
48
What are monoclonal antibodies?
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
What are examples of antigens for immunocytochemical techniques?
- Protiens, glycoproteins, proteoglycans
50
How do we produce a hybridoma?
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
What is direct labeling for antibodies?
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
What is indirect labeling for antibodies?
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
What does it mean when a cell is stained a certain color while stained with (for example: anti-insulin)
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)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions