Histology: Junqueira 13th ed.

 Infiltrated tissue is placed to a paraffin
Jaymee B. Quindara mold at room temp
 Plastic Resin – embedding that avoids
Chapter 1: Histology and Its Methods of Study placing tissues to high temps  no
shrinkage and distortion
I. Overview
 Histology F. Trimming
 Histo (Greek): tissues/ web  Sectioning
 Study of tissue of the body  Microtome
 How tissues are arranged to constitute  1-10 m
organs
 2 interacting components G. Staining
 Cells  Make tissue structure more visible
 ECM
 Consist of macromolecules a. Basic dyes
(collagen, basement membrane)  Toluidine blue, Alcian blue, Methylyn blue
 Support cells & Hematoxylin
 Transport of catabolites and  Stain acidic cellular components w/ (-)
secretory materials charge blue – nucleic acid, DNA, RNA,
 Produced by cells GAGs, Nucleus, cartilage matrix
 Affect cells in their activities  GAGs – anionic unbranched long chains of
 Fxn w/ cell polysaccharides containing aminated sugars
 Tissues formed by cell specific assoc btwn cells &
ECM b. Acidic dyes
 Facilitates recognition via microscopy  Eosin, Orange G, & Acid Fuschin
 Organs formed by combi of several tissues hence  Stain basic cationic components pink –
affecting their fxn CHONs w/ many ionized amino grp,
mitochondria, secretory granules & collagen
II. Preparation of Tissues
A. Fixation c. Hematoxylin & Eosin (H&E)
 Initial step  Hematoxylin stain Basophilic structures
 Remove degrading enzyme and bacteria  Eosin stain Acidophilic structures
 Tissues cut first to allow easy fixative
d. Periodic Acid- Schiff Reagent (PAS)
diffusion
 Transformation of 1-2 glycol grps in sugar
 Fixatives – sol that stabilize and cross links
to aldehyde residue that reacts to Schiff
compound by reacting w/ NH2 (Amine) of
producing Purple-Magenta color
tissue CHONs preventing degradation
 DNA can be stained to be identified &
 Formalin – buffered sol of 37%
quantified in nuceli w/ Feulgen rxn due to
formaldehyde
hydrolysis of deoxyribose by mild HCl
 Glutaraldehyde – reinforce fixation by cross
followed by PAS
linking CHONs via its being a dialdehyde  PAS (+)
 In EM – double fixation; Glutaraldehyde +
 Glycogen – liver, striated muscles
Osmium tetroxide (preserve and stain
GylcoCHONs
membrane lipids & CHONs)
GAGs
B. Dehydration Proteoglycans – GAGs w/ CHON core
 Extraction of H2O  PAS (+) can be further identified by
 Immersion from increasing concentration enzymes
of alcohol (ethanol) 70% to 100%\
e. Counter staining
C. Clearing  Single stain applied separately to allow
 Alcohol removal better recognition of structures
 Replacement of miscible organic solvent to
alcohol & embedding medium f. Lipid Soluble dyes
Toulene  Sudan black
 Preserve lipid rich structures
D. Infiltration
 Infiltration of paraffin in 52oC – 60oC g. Metal Impregnation
 Silver salts
E. Embedding

Electron Microscopy  Flouresence Microscopy  Intrxn of tissue components w/ beams of e- Tissues are irradiated w/ UV light  Wavelength is much shorter than light and emission is in visible spectrum Appears bright in dark BG A. cellulose. smallest distance btwn 2 particles at w/c the can be seen as separate objects 0.voltage difference btwn cathode and abode varies btn visible images from transparent 60-120 kV producing e beams w/ diff wavelength objects . Eyepiece organized subunits Condenser – collects & focuses Polarizing filter Objectives – enlarges & project to Birefringence – ability to roatte the eyepiece direction of vibration of polarized Eye piece – magnifies & projects to light – collagen. eyes microtubules & actin filaments Resolving power – critical factor in getting detailed image.  Common method of visualizing ECM Use plate w/ pinhole infort of fibers and specific nervous tissue image detector Scanning specimen at successive focal planes w/ a focued light beam and produces 3D reconstruction III. Visualizing Specific Molecules Use small point of high intensity IX.applies only to isolated macromolecules or particles actin filaments green .very thin tissue sec magnified at 120. Autoradiography 3D VI. Enzyme Histochemistry  Confocal Microscopy VIII. Use lens system that produce . Scanning Electron Microscope Differential interference microscopy with Nomarski optics – V.beams focused by passing through electromagnets with Living cultures possible variable strength Different speed of light passing through different structures Refractive indices B.magnification up to 400. Transmission Electron Microscope Acridine orange – DNA yellow. Light Microscopy from the images  Bright field Microscopy Ordinary light passing through the  Polarizing Microscopy specimen Allows recognition of stained/ Optical components: Condenser. .2 m IV. Interpretation of Structures in Tissue Sections light often from laser . . 000 x .nucleus blue.permits resolution around 3nm RNA orange . 000 x DAPI & Hoechst. unstained structures made of highly Objectives.metallic filament cathode emits electrons that move toward an anode – metal late w/ central hole that forms a  Phase-Constrast Microscopy beam of electrons passing through it. Cell & Tissue Culture VII.