Histology and Embryology
Programme of the 1st practice
general information
(organization of teaching)
histology and embryology
(what is the subject of study)
tissue processing for the light and electron microscopy (laboratory methods)
demonstration of histological slides
(staining by differenent methods )
Organization issues
Beginning - strictly on time
Change your shoes - you will not be allowed to enter the hall w/o
indoor shoes
Lockers – jackets, coats, bags etc.
Cell phone – switched off or in silent mode
Microscopic hall = laboratory
– eating, drinking, smoking not allowed
– smoking strictly forbidden anywhere in LF
– students have to follow the instructions
– academic misconducts or inappropriate behavior result in excluding
from the lesson or course
Follow safety rules
You have dedicated working place
You are responsible for microscope, slide set, EM atlas
Practical lesson
−introduction; the images free available through Atlas of Histology
−your individual work = study of the slides, schematic but precise drawing of
tissue architecture, careful description. You make your own „study atlas“.
−students come prepared for practices - schedules and syllables – pin-boards
or dpt. webpage
−break – 10 minutes
Attendance
−100% attendance
−substitution only in exceptional cases, after permissions from both the
teacher of your group and the lesson where you plan to substitute
−sign in to the list
−make a protocol, let it check and signed by the lecturer
Registration of substitution:
Datum Jméno Ročník Skupina Č. praktika Č. místa Vyučující - podpis
Date Name Year Group Nr. of practice Nr. of place Teacher- signature
• Protocols
− you have to make paper protocols (no tablets, laptops)
− A4 size, blank, without lines, according to the template (can be downloaded
from www.med.muni.cz/histology - Education)
− (color)pencil handdrawings (no pen)
− complete set of signed protocols is required for getting the credits
− the quality of the protocol is approved by your teacher’s signature at the end
of practical lesson
− incomplete or low-quality protocols cannot be approved and you have to
substitute the respective practical lesson
Credits
−100% attendance
−complete set of signed protocols from all lessons
−credit test
•the student must pass a credit test
•the test will be organized as a ROPOT in the IS
•the test will open in the last 14 days of regular teaching (prior dissections)
•number of correct answers to successful pass the test is 90%
•there is unlimited number of attempts until the test is closed, only the best score will be
recorded.
Absence at the practicals
It is mandatory for the student to:
In the earliest possible term inform her/his teacher and at the same time provide official
valid apology to the Study department (International Office). The excuse must appear in
the IS. Specific situations can be approached individually.
Substitute the given practical.
Substitution of the practical can be achieved via two routes:
In presence: student will attend the practical as agreed by the teacher and will produce
the standard protocol.
At distance: if the “in presence” substitution is not possible because of specific
conditions, student will 1) produce the complete protocol based on the materials available
online (Atlas of Histology, Atlas of Cytology and Embryology), 2) elaborate answers to set
of questions (available in the IS) and 3) online consult key elements of the practical.
The preferred form of substitution is „in presence“.
End of practical lesson:
−the practice is closed by the lecturer
−you are allowed to leave your working place only after checking the
microscope and slides
−if you leave before the check you may be responsible for any
damages/losses recognized later
During the semester, the primary contact is the teacher of student’s
study group. Name of the teacher can be found in the Timetable, the
email contact then in the IS.
Alternatively, a general email contact can be used:
histology@med.muni.cz
http://www.med.muni.cz/histology
Department of Histology and Embryology
Faculty of Medicine MU
Head: prof. Aleš Hampl
RECOMMENDED LITERATURE
Mescher, A.L. Junqueira's basic histology :text and atlas. 13th ed. New York: McGraw-Hill Medical, 2013. xi, 544.
ISBN 9781259072321.
Moore, K.L., T.V.N. Persaud a M.G. Torchia. The developing human: clinically oriented embryology. 9th ed. Philadelphia,
PA: Saunders/Elsevier, 2013. xix, 540. ISBN 9781437720020.
Ovalle, W.K., P.C. Nahirney a F.H. Netter. Netter's essential histology. 2nd ed. Philadelphia, PA: Elsevier/Saunders, 2013.
xv, 517. ISBN 9781455706310.
Young, B. Wheater's functional histology :a text and colour atlas. 5th ed. [Oxford]: Churchill Livingstone, 2006. x, 437.
ISBN 044306850X.
Sadler, T.W. a J. Langman. Langman's medical embryology. Illustrated by Jill Leland. 11th ed. Baltimore, Md.: Lippincott
William & Wilkins, 2010. ix, 385. ISBN 9781605476568.
Lowe, J.S. a P.G. Anderson. Stevens and Lowe´s Human Histology. 4th. : Elsevier, 2015. ISBN 978-0-7234-3502-0.
Lectures
Protocols
RECOMMENDED LITERATURE
Department of
Histology and
Embryology MF MU
http://www.med.muni.cz/histology
http://www.med.muni.cz/histology/multimedia-and-textbooks/
HISTOLOGY
structure and ultrastructure of normal cells and tissues,
cytology and general histology
special histology = microscopic anatomy of individual organs
relevance: oncology, surgery, hematology, pathology,
forensic,…
EMBRYOLOGY
– prenatal (intrauterine) development
General embryology (until 2nd month – EMBRYO )
gametogenesis and early embryonic development
Special embryology (since 3rd month to birth – FETUS )
organogenesis
Teratology – defects in organ development, malformations, anomalies;
prenatal screening – ultrasonography, amniocentesis, genetic and karyotype
screening
Relevance: gynecology and obstetrics, pediatrics, assisted reproduction
Histology
and
ulstrastruc
tural
architectur
e
Cell
biology
Molecular
biology
Physiolog
y
Biochemis
try
Gene, cell
and tissue
engineerin
g
Quantitative analyses
of cells and tissues
- Genomics
- Transcriptomics
- Proteomics
- Metabolomics
- …
Understaning of the
whole system
- Personalized medicine
- Regenerative medicne
- Assisted reproduction
- Gene therapy
- Cancer therapy
- Biomedical research
Histology cannot be put out of the biological and functional context
Histology
•Resolution of naked eye – 0.1 mm
•Resolution of light microscopy – 0.1- 0.5 m
•Resolution of electron microscopy – 0.1 - 1 nm
Tissue processing for the light microscopy (LM)
(making of permanent preparations – slides)
SAMPLING (obtaining of material – cells, tissue pieces)
FIXATION of samples (tissue blocks)
RINSING (washing) of samples
EMBEDDING of samples - embedded blocks
CUTTING of blocks - sections
AFFIXING of sections
STAINING of sections
MOUNTING of sections
SAMPLING
A small piece of organ (tissue) is sampled and quickly put into the
fixative medium.
Biopsy during surgical dissection of organs in living organism
= excision
= puncture (liver or kidney parenchyma, bone marrow)
= curettage (uterine endometrium, adenoid vegetation)
Necropsy from dead individual (sections); in experiments laboratory
animals are used and tissue have to be sampled as soon as possible
after the break of blood circulation
The specimens shouldn't be more than 5 – 10 mm3 thick and fixation
should follow immediately.
Aids to sampling:
FIXATION
Definition: denaturation and stabilization of cell proteins with minimum artifacts
The purpose of fixation: freshly removed tissues are chemically unstable – dry,
shrink, undergo hypoxia, autolysis and bacteriological changes
To stop or prevent these changes and preserve the structure tissue samples
have to be fixed. During the fixation, all tissue proteins are converted into
inactive denaturized (stable) form.
3 main requirements on fixatives:
- good preservation of structure
- quick penetration into tissue block
- no negative effects on tissue staining
Fixatives: solutions of different chemicals
- organic fixatives – ALDEHYDES – formaldehyde (most frequently used for
LM)
– glutaraldehyde (used for EM)
– ALCOHOLS – 96 – 100 % (absolute) ethylalcohol
– ORGANIC ACIDS – glacial acetic acid, picric acid,
trichloracetic acid
- inorganic fixatives – INORGANIC ACIDS – chromic acid, osmium tetraoxide
(OsO4)
– SALTS OF HEAVY METALS – mercuric chloride HgCl2
- compound fixatives – mixtures (two or more chemical components to offset
undesirable effects fo indiviual (simple) fixatives.
FLEMMING´s fluid – with OsO4
ZENKER´s and HELLY´s fluid, SUSA fluid – with HgCl2
BOUIN´s fluid – with picric acid
CARNOY´s fluid – with alcohol
Fixation is carried out at the room temperature, the time varies between 12 – 24
hours, specimen must be overlayed by 20 – 50 times fixative volume:
Ratio of tissue block volume to fixative volume 1 cm3 : 20 – 50 cm3
RINSING and EMBEDDING
All samples should be washed to remove the excess of
fixative; the choice of rinsing medium is determined by type
of fixative: running tap-water or 70-80% ethanol
Relevance of embedding: tissues and organs are brittle and
unequal in density, they must be hardened before cutting
Embedding media
▪water soluble – gelatine, celodal, water soluble waxes
▪anhydrous – paraffin, celoidin
EMBEDDING into PARAFFIN
• dehydration – to remove water from fixed samples by
ascending series of ethanol is used (50%, 70%, 90%,
96%. each step - 2 – 6 hours
• clearing – the ethanol must be replaced with organic
solvatant that dissolves paraffin – xylene
• infiltration – melted paraffin wax (56°C) is used; 3 x 6
hours.
• casting (blocking out) – moulds (plastic, paper or metal
chambers) are used for embedding
The moulds are filled with melted paraffin, tissue samples are then
placed inside and immediately immersed in cold water to cool paraffin
quickly down.
These paraffin blocks are ready for trimming.
Automated device for tissue dehydration
Leica TP 1020
Paper chambers
- metal
CUTTING
Microtome – a machine with automatic regulation of section thickness:
5 – 10 μm is optimum.
sliding microtome – block is fixed
in holder, knife or razor moves
horizontally
rotary microtome – knife is fixed,
block holder moves vertically
Rotary microtome
Sliding microtome
Freezing microtome (cryostat)
= rotary microtome housed in freezing box
(– 60º C)
Cutting of frozen tissue without the embedding
CUTTING
AFFIXING
Mixture of glycerin and egg albumin or
gelatin
Section are transferred from microtome
razor or knife on the level of warm water
(45º C), where they are stretched; then
they are put on slides coated with
adhesive mixture; excess of water is
drained and slides are put in incubator
(thermostat, 37º C) over night to affixing
of sections.
1 2 3
4 5 6
8
sampling fixation fixation
cutting cutting
section affixing section affixing
embedding
7
Stretching of sections on warm water
Stretching on a warm plate
STAINING
•Different cell or tissue structures are not apparent without
staining.
•Cellular structures exhibit different affinity to staining dyes:
alkaline dyes (basic or nuclear) – react with anionic groups of
cell and tissue components
basophilia – basophilic structures in the cell
acid dyes (cytoplasmic) – react with cationic groups
acidophilia – acidophilic structures in the cell
neutrophilia – no reaction
Hematoxyline and eosin (HE)
cell
cytoplasm
cell nuclei
collagenous
connective
tissue
xylen I xylen II 100% 96% H2O hematoxyline acid
ethanol ethanol ethanol
xylen IV xylen III 100% 96% H2O eosin H2O
ethanol ethanol
HEMATOXYLINE – EOSIN (HE)
Deparaffination Rehydration Washing Staining Differentiation
Clearing Dehydration Washing Staining Washing
ROUTINE STAINING with HEMATOXYLINE –
EOSIN (HE)
Hematoxyline – basic (nuclear) dye
Eosin – acid (cytoplasmic dye
Staining procedure:
paraffin must be removed (dissolved) by xylene
sections are rehydrated in descending series of ethanol (100% 96%
80%)
staining with hematoxyline
differentiation in acid ethanol and water (excess of dye is removed)
staining with eosin
rinsing in water (excess of dye is removed)
dehydration in graded ethanol series (80% 96% 100%)
clearing in xylene
Staining results:
HE = Hematoxyline – Eosin
nuclei – bright clear blue or dark violet
cytoplasm and collagen fibers – pink
muscle tissue – red
HES = Hematoxyline – Eosin – Safron
connective tissue – yellow
AZAN = AZocarmin – ANiline blue – orange G
nuclei – red
erythrocytes – orange
muscle – red
collagen fibers – blue
cuvette
flask
slides holder
(basket)
Staining tools:
Automatic slide stainer
staining set of boxes with
media
MOUNTING
•Finally, preparates are closed with coverslip (coverglass) to form a
permanent preparate. Small amount of mounting medium must be placed
between stained section and the coverslip.
•Mounting media: soluble in xylene – canada balsam
soluble in water – glycerin-gelatine, arabic gum
Permanent histological slides for study in the light microscope
Hematoxyline and eosin (HE)
Azocarmine and aniline blue (AZAN)
collagen fibers are blue
ren
Staining methods:
routine – HE, AZAN
(demonstrate all components of tissue)
special
visualizes only special structures
impregnation
by silver salt for detection
of nerve or reticular fibers
HE – the most frequent used method
lipid droplets
detected by oil red
Histochemistry and Immunohistochemistry
•Relevance:
various chemical compounds detected „in situ“ (proteins, AA,
NA, saccharides, lipids, enzymes, pigments, inorganic
substances – Fe, Ca, Zn)
Various epitopes detected by immunotechniques.
Antigen
Primary Ab specific
against epitope of the
particular antigen
Secondary Ab specific
against primary Ab
Enzyme conjugated with
secondary Ab - visualization
Actin
(cytoskeleton)
DAPI (nucleus)
Microtubules
(cytoskeleton)
KI-67
In-vivo/live cell imaging
- US, MRI, PET…
- cells with fluorescent reporter
doi:10.7150/thno.3694
FACS
Tissue processing for the EM
• pH of all solutions (media) must be buffered on 7.2 – 7.4
Cacodylate or Phosphate buffer is frequently used.
• Absolutely dustfree environment
• Solutions (media) have to be precise (artifacts)
Tissue processing for the EM
SAMPLING – immediatelly after arresting of blood circulation, tissue block
sized no more than 1mm3
FIXATION – glutaraldehyde (binds amine groups) + OsO4 (binds lipids) are
used as double fixation
RINSING – distilled water
DEHYDRATION - ethanol
EMBEDDING – gelatin capsule or plastic forms are filled with some medium
(which can be polymerized from liquid to solid form) and pieces of fixed
tissue are placed into this medium. Epoxyd resins (Epon, Durcupan, Araldite)
are usually used as in water insoluble media.
CUTTING – ultrathin sections (in ultramictomes)
CONTRASTING ≈ staining
Embedding tools:
gelatin (1) or plastic (2)
capsules
capsule holder (3)
embedding plates
(4, 5)
Embedded blocks
prepared for cutting
1 2
3
4,5
By trimming, using ultramicrotome, an
excess of hard medium is removed and
pyramide with minimal cut surface
(0.1 mm2) is prepared.
Minimum of tissue (black) is in the top
of pyramid
Cutting
Ultrathin sections (70 – 100 nm) -
ultramicrotomes.
Glass or diamond knives with water
reservoir are used
Sections slide flow on water in
small container attached to the
knive
Supporting grids
Ultramicrotom
knives:
glass
diamond
Cutting
CONTRASTING (=STAINING)
• principle of differentiation of structures – different dispersion of
beam of electrons depending on atomic weight of elements.
„electron dyes“ are thus mixtures of heavy metals: uranylacetate or
lead citrate
stain droplet with floating grid
placed section-side down on the
droplet
Differences between LM and EM
LM EM
Sampling  1 cm3
minutes
 1 mm3
seconds
Fixation formaldehyde
12 – 24 hours
glutaraldehyde
1 – 3 hours
Embedding paraffin epoxid resins (Durcupan)
Cutting
Thickness of sections
microtome
5 – 10 m
Ultramicrotomes
50 – 100 nm
Staining (LM)
contrasting (EM)
dyes
(hematoxyline – eosin)
heavy metals
(uranylacetate,lead citrate)
Mounting (only LM) ---
Result histological slide (preparate) photograph of ultrathin section
Visit us at:
http://www.med.muni.cz/histology
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