Methods in Cell Biology

Medical Microanatomy 602

Edie C. Goldsmith, Ph.D.

Edie.Goldsmith@uscmed.sc.edu

Introduction

• The material in this power point is covered in Chapter 1 of your textbook. I would encourage you to read the text, particularly sections that are covered here.

• While we will not specifically cover this material during lecture, you will be expected to know this information for the exam. I can address any questions you may have during the lab period or by email.

• Notice that in the notes section below each slide I have added information and explanations for the images/text on that slide when necessary.

Tissue Preparation• Fixation

– Preserve tissue structure– Chemical

• Formaldehyde/glutaraldehyde - cross-linking amine groups• Alcohol – denature proteins• Heavy metals (osmium tetraoxide) – oxidation of proteins to

form cross-links; also react well with phospholipids– Freezing

• Cryoprotectant (OCT, sucrose solution)• Rapid low-temp freezing• Lipids and activity stains (enzymes)

• Not all fixative works for all structures– Formaldehyde - not good for lipids

• Don’t over fix

• Dehydration & Clearing– Prepare tissue for embedding– Removal of water can cause tissue shrinkage

• Infiltration– Mix of clearing agent and embedding media

• Embed tissue– Prevents tissue damage during sectioning– Embedding agents - Paraffin, plastic resins,

acrylamide

• Section, mount and stain

• Samples are dynamic during processing

• Some things get retained– Nucleic acid – protein complexes, cytoskeletal proteins,

ECM proteins, lipid-protein complexes

• Some things get lost– Small, soluble molecules (ions, carbohydrates), glycogen,

proteoglycans– Can use special fixation or staining methods

Tissue Staining

Basophilic

• Stain with basic dye [dye+Cl-]

• Toluidine blue, methylene blue, hematoxylin, alcian blue

• Nucleic acids, some cytoplasmic components (rRNA and rER), glycosaminoglycans and acidic glycoproteins

Acidophilic

• Stain with acidic dye [Na+dye-]

• Orange G, eosin, acid fuschin

• Mitochondria, cytoplasm, secretory granules, ECM proteins

Dye color does not determine whether a dye is acidic or basic(see Table 1.2 in your textbook)

• Hematoxylin & eosin (H&E) is the most common dye combination you will see this semester.– Hematoxylin – basic-

like dye which stains negatively charged molecules (blue)

• i.e. Nucleic acids

– Eosin - acidic dye which stains positively charged molecules (pink)

• Cytoplasm (proteins)

Material stained with eosin

Nuclei stained withhematoxylin

Metachromasia

• When certain dyes bind some cellular structures, their color shifts

• This is due to the presence of a large number of polyatomic anions (SO4

2- and PO43-)

– This leads to aggregation of the dye which changes is absorption properties

– The polyatomic anions are common in ground substance (particularly cartilage); heparin granules in mast cells; rER in plasma cells

• Toluidine blue is an example of a dye the exhibits metachromasia. As it name would imply, this dye usually stains blue. But when it is used to stain mast cells, the granules in these cells contain lots of negatively charged molecules and the dye’s color shifts to a purple-red appearance.

Other notable stains

• Periodic acid-Schiff (PAS)– Carbohydrates (glycogen,

mucin, basement membrane)

– Periodic acid + sugar

aldehyde

– aldehyde + bleached basic fuchsin (Schiff reagent)

magenta color

PAS positive Staining (mucus)

Orcein

• Also known as resorcin-fuchsin

• stains elastin found in elastic fibers

• The individual fibers in this image are elastic fibers stained with orcein

Aorta

• Trichrome stains – Employ 3 dyes– Masson Trichrome– most

common– Usually used to look at

collagen in the extracellular matrix

• Nuclei (basophilic) – blue/black

• Collagen – green or blue• Cytoplasm, muscle, keratin,

erythrocytes – red

• Azan– Stains collagen, basement

membrane, mucin• Nuclei – red• Collagen - blue

nephroncollagen

nucleus

collagen

nucleuserythrocytes

Picrosirius Red

• Collagen specific stain• Observed under

polarized light the collagen appears yellowish-orange in color.

• Observed under normal bright field microscopy, the collagen would have a bright pink appearance. In this image of skeletal muscle, the collagen has

a bright, glowing orange appearance. The striatedstructures are muscle cells.

Nissl

• This uses a basic dye on neuronal tissue.

• Stains the rER in neurons– Referred to as Nissl

substance when observed in clumps

• Can also be done with methylene blue

N

Gold and Silver Stains

Silver stain of reticular fibers(type III collagen)

String-like appearance in this image

Gold staining of motor end platein muscle

Renal cortex

Lipids

• Preserved in frozen sections

• Stains used in light microscopy– Sudan black, Oil red O,

Sudan IV, Osmium tetraoxide

• Common stain used for electron microscopy– Osmium tetraoxide

Black circles in this image are lipiddroplets in brown fat cells

Enzyme Histochemistry

• Localize enzymes within tissue

• Don’t over fix (frozen sections)

• Precipitated product

• Indirect method

• Variety of enzymes– Acid and alkaline

phosphatases, dehydrogenases, peroxidases

• Light and EM Alkaline Phosphatase in rat kidney(Gomori stain with a lead precipitate)

Immunohistochemistry (IHC)

• Rely on the use of antibody directed against molecule of interest– Usually protein

• Direct vs indirect• Amenable to many

forms of detection– Fluorescence– EM (gold)– Light microscopy (color

precipitate)Blue material in this fluorescent image is acell surface protein that binds collagen.

Orange indicates sites of peroxidase reaction(lysozyme in small intestine)

-amylase ab stained with Protein A - gold

In Situ Hybridization

• DNA or RNA distribution

• Target specific nucleotide probe– Radioisotope, biotin,

digoxigenin

• Detected by autoradiography, color precipitate, fluorescence

Parathyroid hormone staining (red-ish color) inParathyroid gland

Fluorescence Microscopy• Light source (UV or laser)

• Dyes which when excited emit in the visible region of the spectrum

• Confocal microscopy– Laser light source– Scan across sample and optically section– Useful on live samples (monitor real time events)– Non-destructive (in that it can be used on live cells)

• Easy sample preparation, fast, inexpensive

Not all fluorescent stains require an antibody

Phalloidin stainingof actin

Acridine orangeDNA – yellow; RNA - orange

Autoradiography

• Rely on the use of radioactive tag to follow molecule– 32P, 35S, 3H

• Radioactive probe (amino acids, nucleotides)• Specimen must be alive to monitor metabolic events• Can be used with other stains• Slide dipped in photographic emulsion & developed• Silver grains where radioactive material is present in

sample (AgBr Ag (precipitate))• Semi-quantitative

Electron Microscopy

• Electron beam as source ( ~ 0.005 nm) • Operate under vacuum• Electromagnets instead of glass lenses• Detect by fluorescent screen or photographic

emulsion• Destructive• Two major types

– Transmission (TEM) and scanning (SEM)

TEM

Cellular ultrastructureBright portion – e- pass throughDark portion – e- absorbed

or scatteredSections – 50 – 150 nmFix – glutaraldehydeStains (e- dense; heavy metals)

Osmium tetroxide (OsO4) – lipidsUranyl acetate, Lead citrate –

non-specific (surface adsorbed)

Ruthenium red – complex carbohydrates

• Surface ultrastructure• Fixed, dried, coated with

gold before imaged• e- reflected from surface

– Results in 3-D like image

SEM

SEM image TEM image

Interpreting EMs

• Use internal rulers as guides– Plasma membrane ~ 10 nm wide– Nucleus between 3-10 m– Ribosomes between 15-30 nm

• Unique organelle features– Mitochondria and Golgi

• Look for largest objects first

Problems & Pitfalls

• Shrinkage – fixation and embedding– Appearance of empty spaces

• Empty spaces due to loss of material– Improper fixation and dehydration

• Wrinkling• Precipitation of stains (may appear as dark spots all

over the section)• Do not memorize images - learn morphological criteria• Do not rely on color

Millette’s Laws

• Law #1 – In any given image, if two objects look the same they are the same.

• Law #2 – In any given image, if two objects look different they are different.

H&E

Goblet cells in Intestine

PAS

A Tale of Two H&E Stains

2-dimensional appearance vs.

3-dimensional structure