LEC 4: Visualizing Cells and Tissues: Stains - 08.18.2014 Flashcards Preview

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Flashcards in LEC 4: Visualizing Cells and Tissues: Stains - 08.18.2014 Deck (51):
1

Hematoxylin

  • acts as a basic (positive) stain
  • stains deep blue-to-purple color
  • positively charged dye so binds to acidic (negative)
  • basophilic structures (structures stained display basophilia)

2

Cell structures that are basophilic (bind with hematoxylin)

  • DNA
  • RNA
  • Heterochromatin and euchromatin
  • nuclei
  • nucleoli
  • ribosomal particles
  • RER

3

Eosin

  • stains pink-red-orange color
  • acid stain (negatively charged dye)
  • binds to basic (positively charged) structures
  • structures stained display acidophilia (eosinophilia)

4

Cell structures that are acidophilic (bind with eosin)

  • cytoplasmic proteins
  • mitochondria
  • filaments
  • smooth ER
  • secretory proteinaceous granules

5

Under what circumstances would the cytoplasm be more blueish

when cytoplasm contains lots of RER (e.g. plasma cells) or free ribosomes (rRNA), cytoplasm will be more blueish

6

What is the difference between euchromatin and heterochromatin

  • euchromatin, DNA is "loose" for active transcription of RNA
  • in heterochromatin, DNA is condensed, inactive transcription of RNA

7

What does the nucleolus do

  • made up of proteins, RNA
  • transcribes and modify rRNA, integrate ribosomal proteins to form immature ribosomes

8

What does the rough endoplasmic reticulum do (RER)

ribosomes and rRNA making up the RER are charged with secretory protein synthesis

9

Free ribosomes

cytosolic protein synthesis

10

What does the smooth ER do

  • lipid synthesis
  • alcohol and drug metabolism
  • glycogen storage

11

What does the Golgi apparatus do

  • carbohydrate synthesis and secretion

12

What does the Mitochondria do

ATP production

13

What do microtubules do

  • movement of molecules, motility
  • mitosis

14

What do filaments do

support, contraction

15

What are the functions of lipid droplets and glycogen in the cell

energy sources

16

What are the major structures in this image

Q image thumb

Hepatocytes (liver cells)

  • nucleus is basophilic
  • nucleolus and heterochromatin are very dark
  • euchromatin is not well stained
  • cytoplasm is pink (mitochondria, SER, filaments)
  • RER is basophilic
  • Golgi, lysosomes, glycogen, and fat are lightly stained or not stained

17

In this image of a plasma cell, what do the arrows point to

Q image thumb

A. RER in cytoplasm is basophilic

B. Golgi apparatus is not stained well, appears as pale/light pink area just off nucleus (hof area)

18

Which two (2) organelles are involved in the synthesis of antibodies (glycoproteins)

Golgi and RER

19

histochemistry

  • chemical methods for detecting chemical make-up
  • identify specific molecules in cells and tissues
  • histochemical methods are also colorful (like H&E stains) but more specific

20

Feulgen Reaction ("foil-gun")

  • for nuclear DNA 
  • mild acid hyrolysis to remove nucleotide from deoxyribose
  • forms aldehyde group with can react with Schiff reagent (magenta color complex)

A image thumb
21

What are the cell phases in this garlic root

Q image thumb

Feulgen reaction staining nuclear DNA

A. Interphase (nuclear membrane/envelope, euchromatin, heterochromatin, and nucleoli are present)

B. Prophase (coarse clumps of heterochromatin signify that chromatin is condensing, continues until end of prophase when nucleolus and nuclear envelope have disappeared)

C. Anaphase (chromatids separate from each other and move toward opposite poles of the mitotic spindle)

22

Periodic Acid-Schiff (PAS) Reaction

histological reaction that detects carbohydrate-rich macromolecules

  • mucus
  • glycosaminoglycans (GAGs)
  • glycoproteins
  • glycogen

A image thumb
23

PAS stain of intestinal epithelium

Q image thumb

  • mucus of goblet cells stained deep red
  • glycoprotein of glycocalyx
  • basement membrane (heparan sulfate = GAG)

NB: These are all carbohydrate-rich macromolecules, which is why they appear in PAS stain

24

What is the difference between the PAS stain of liver cells (hepatocytes) and the unstained?

Q image thumb

diastase was used as a control to digest glycogen before PAS stain applied (no carbohydrates to stain)

25

diastase

a control method; enzyme digests glycogen prior to PAS staining

26

metachromasia

staining reaction in which the cell components are stained a different color from the dye color

27

Toluidine Blue

  • cationic dye (positively charged)
  • metachromatic property
  • deep blue color
  • stains heparin in mast cells (highly sulfated GAG)

28

H&E stain of Heparin in mast cells

vs. 

Toluidine (tah-luah-deen) blue stain of Heparin in mast cells

Q image thumb

Heparin in mast cells stains acidophilic with H&E (pink)

vs. 

Heparin in mast cells stains magenta with toluidine (metachromatic reaction)

29

Structure of Heparin

Q image thumb

  • Heparin is a glycosaminoglycan (GAG)
  • repated disaccharide units
  • glucosamine and uronic acid
  • residues are sulfated, so HIGHLY NEGATIVELY charged

30

Sudan IV

Q image thumb

Fat stain

  • oil soluble dye with a deep red color

31

Frozen section of adipose tissue, what stain is used

Q image thumb

Sudan IV stains fat in adipocytes

32

Osmium tetroxide (OsO4) Stain

Lipid Stain, strong oxidizing compound 

33

In this slide of myelinated axons, what stain is being used

Q image thumb

OsO4 is staining the fatty (lipid) myelin of the axons black. 

  • Note Node of Ranvier where myelin sheath is not present

34

Resorcin-fuchsin Stain (re-source-in fuuksin)

stains elastic fibers in extracellular matrix black

35

H&E Stain of elastic fibers

vs.

Resorcin-fuchsin Stain of elastic fibers

Q image thumb

  • H&E Stain, elastic fibers are acidophilic (pink)
  • Resorcin-fuchsin Stain, elastic fibers stain black

36

Mallory Trichrome Stain

  • more sensitive than H&E
  • routinely used in clinics for histopathological diagnosis of disease related to fibrosis
    • hepatic fibrosis
    • pulmonary fibrosis

NB: fibrosis = formation of excess fibrous connective tissue in organ/tissue in a reparative or reactive process

  • hepatic fibrosis = excessive connective tissue builds up in liver (can lead to hypertension, scarring distorts liver blood flow, or cirrhosis)
  • pulmonary fibrosis = excessive connective tissue in lungs hinders lung function (shortness of breath)

37

H&E Stain of collagen fibers in connective tissue

vs.

Mallory Trichrome Stain of collage fibers (Type I and III) in connective tissue

Q image thumb

  • collagen fibers are acidophilic (pink) in H&E Stain
  • collagen fibers (Type I and III) stained blue in Mallory Trichrome Stain

38

Silver stain

Stains Type III collagen (a.k.a. reticular fibers) black

39

Stain of Type III collagen (reticular fibers) in spleen

Q image thumb

reticular fibers are stained black with silver stain

40

enzyme histochemistry

permits localization of enzyme activity in a tissue section using:

1. substrate of enzyme

2. capturing agent (will combine with enzyme product to form insoluble precipitate, visible)

41

Acid phosphatase

marker enzyme for lysosomes

42

Azo dye method

  • Uses diazonium as capturing reagent for acid phosphatase
  • carried out at pH=5
  • produces a Diazonium complex that appears RED

A image thumb
43

Kidney tubule cells treated with Azo dye method

Q image thumb

  • Azo dye method produces a Diazonium that appears RED
  • localizes acid phosphatase
  • presence of lysosomes

 

44

Succinate dehydrogenase (SDH)

oxidate enzyme of the citric acid cycle (TCA), localized in mitochondria (marker of mitochondria)

45

Tetrazolium method

  • uses Tetrazolium as capturing reagent
  • produces insoluable black Formazan under physiological temperature and pH

A image thumb
46

Submandibular gland treated with Tetrazolium method

Q image thumb

  • SDH found in ducts (black appearance)
  • indicates presence of mitochondria

 

47

immunohistochemistry

permits detection of proteins in cells or tissue sections based on reaction between antigen and its antibody

48

What are the two (2) types of immunohistochemical methods

1. antibody marked with fluorescent compound

2. antibody marked with peroxidase

A image thumb
49

Diaminobenzidine (DAB) method

  • demonstrates peroxidase activity
  • creates insoluble brown color compound
  • DAB oxidized to insoluble brown compound at site of peroxidase activity

50

Actin filaments (2 different treatments)

Q image thumb

L - detection of actin filaments with actin antibody (antibody for actin protein) labeled with fluorescent dye

 

R - detection of actin filaments with actin antibody labeled with peroxidase (DAB method)

 

NB: DAB method oxidized to insoluble brown compound at site of peroxidase activity

 

51

Immunoperoxidase staining for insulin in islet of Langerhans of pancreas

Q image thumb

  • insulin is a peptide hormone
  • antibodies for insulin tagged with peroxidase
  • DAB (diaminobenzidine) oxidized to insoluble compound at site of peroxidase activity (brown color)