Modern Immunofluorescence Techniques
Lecture 9
Tissue sectioning for immunofluorescence
microscopy
Preparation Schemes
• Cell Monolayers
• Cell Suspensions and unicellular organisms
• Tissue sections
• Wholemounts
Preparation Schemes
• Cell Monolayers
• Cell Suspensions and unicellular organisms
• Tissue sections
• Wholemounts
Preparation Schemes
• Sections are made from bulky and compact
organs that cannot be processed per se
• Samples are fixed and embed in solid matrices
that can be cutoff with ease and precision
Preparation Schemes
• In wholemount preparations tissue integrity is maintained. This is important to uncover intercellular spatial relationships
• After sectioning integrity of the tissues is not maintained, but from serial sections it could be reconstructed.
• In wholemount the outer boundaries of the specimen (e.g., cell walls in plants and ECM in animal samples) must be disrupted to allow antibody penetration
• In sections internal surfaces of cells are opened and thus better accesible for antibodies.
Preparation Schemes for wholemounts
Grow material in culture medium
Fix in RT FA for 1 hr in vacuo and wash
Digest cell walls with enzyme cocktail 20 min, RT and wash*
Extract membranes with detergent for 1 hr RT and wash
Block with BSA for min 1 hr RT (also O/N)
Primary antibody (1 day RT)/wash (at least 2 hours)
Block residual aldehydes with NaBH4 15 min RT and wash*
3 hrs 37oC then
O/N at 4oC
Counterstain
with DAPI
Mount in PDA
Secondary antibody/wash (2 hours)
Preparation Scheme for Sections Grow cells in culture medium
Fix cells in RT FA for 30 min
Wash in buffer for max 30 min Dehydrate with solvent
(will take several hours)
Block for 1 h
Primary antibody/wash
(2 hours to overnight depending on cell size)
Secondary antibody/wash (2 hours) Mount on derivatized coverslips
Infiltrate with solid matrix
(e.g., Steedman wax, parafin, paraplast etc) No needs for cell wall
enzymatic digestion
No needs for plasma membrane
detergent permeabilisation
Fixation is the most important step in performing histologic
specimen preparation techniques.
• Poorly fixed specimens are almost always more difficult to section
than those that are well fixed
• Poorly fixed tissue will always produce inferior morphology even if
optimally processed and carefully sectioned
Embed Specimens Carefully
• Embedding is an important step that requires a thoughtful approach.
• Careless embedding can make microtomy much more difficult.
Embedding
Process by which cells, tissues or organs are surrounded by a medium
such as agar, gelatin, wax or plastic which when solidified will provide
sufficient external (and internal) protection during sectioning.
Commercial paraffin wax • Policrystalline mixture of solid hydrocarbons produced during the refining of coals
and mineral oils.
• Usually it is a mixture of straight chain or n-alkanes with a carbon chain length of
between 20 and 40
• The wax is a solid at room temperature but melts at temperatures up to about 65°C
or 70°C.
• Paraffin wax can be purchased with melting points at different temperatures, the
most common for histological use being about 56°C–58°C, but available are with
melting points from 39°C to 68°C.
• Properties of paraffin wax are improved for histological purposes for :
- improving of ribboning
- increasing hardness
- improving adhesion between specimen and wax
- decreasing melting point
Embedding
Trimming the paraffin block using a scalpel
Paraffin tables made from hardwood
Mounting the paraffin block on a piece of hardwood using a heated hobby knife
Mounted paraffin block on a piece of hardwood and rotary microtome
Microtome
Microtome
Microtome
Set Blade Clearance Angle Optimally
• Blade clearance angle is adjustable and must be set for optimum performance
• For most of the knifes and blade holders a setting of between 1° and 5° is
recommended
Left: clearance angle too obtuse. The knife will scrape a section from the block rather than cutting one. There
will be very noticeable cutting artifacts. In extreme cases (as in the sketch) the specimen will scatter
Middle: clearance angle okay, about 5°-10°, slightly more or less depending on specimen, paraffin,
temperature etc…
Right: clearance angle too acute: The paraffin block will be crushed against the rear cutting facet of the
microtome knife and the paraffin table
Cross-sectioned view of microtome knife type C showing the cutting facet‛s angle
Photographs showing the paraffin blocks with samples, cut with a rotary microtome, and the ribbons
of sections extending over the knife.
A) Blocks of material submitted to vacuum during paraffin embedding.
B) Control not submitted to vacuum during paraffin embedding.
Sectioning
Consider Factors Affecting Section Thickness The choice of slide and adhesive will be influenced by the staining methods to be subsequently
applied
Sectioning
Left: curved ribbon due to inadequate trimming: upper and lower side of the block are
not parallel.
Middle: block trimmed okay
Right: left side of block trimmed at an angle to distinguish the sections.
Sectioning
Clearance angle too acute. Left: first partial sections. Middle: thin-and-thick sections. Right: crushed paraffin
at the upper right corner and right side of the block
Orientate Specimen Appropriately The orientation of the specimen to the blade during the cutting stroke can affect the ease with
which a ribbon can be obtained and directly influence section quality
Sectioning
Some of the most common faults seen in paraffin sections are:
Some of the most common faults seen in paraffin sections are:
Some of the most common faults seen in paraffin sections are:
Some of the most common faults seen in paraffin sections are:
Some of the most common faults seen in paraffin sections are:
Some of the most common faults seen in paraffin sections are:
Once fixed, tissue is processed, using gentle agitation,
usually on a tissue processor, as follows:
• 70% ethanol for 1 hour.
• 95% ethanol (95% ethanol/5% methanol) for 1 hour.
• First absolute ethanol for 1 hour .
• Second absolute ethanol 1½ hours .
• Third absolute ethanol 1½ hours.
• Fourth absolute ethanol 2 hour.
• First clearing agent ( Xylene or substitute) 1 hour.
• Second First clearing agent (Xylene or substitute) 1 hour.
• First wax (Paraplast X-tra) at 58°C for 1 hour.
• Second wax (Paraplast X-tra) at 58°C 1 hour.
Infiltration with paraffin
• “clearing” - in the old days the samples were, after
complete dehydration, brought in a solvent of paraffin such
as xylene, benzene or toluene.
• These hydrocarbons with a high refracting index (making
the samples more or less translucent or transparent, thus the
name “clearing”), are rather nasty (poisonous,
carcinogenic, causing liver damage and so on), so
substitutes were sought and found.
Steedman’s wax
Embedding medium, prepared by mixing polyethylene glycol (PEG) 400 distearate with
1-hexadecanol in 9:1 (w/w). Alternatively, the complete medium is commercially
available as Polyester wax. The medium is used at 37 °C.
• Suitable embedding medium for immunohistochemistry, which can effectively
protect protein antigenicity during processing.
• Introduced to histochemistry in 1957.
• Ribbon-forming sectioning medium has several advantages making it for a perfect
tool in the indirect immunofluorescence detection of proteins.
• Low melting-point polyester wax has a melting point of 35–37 °C and is soluble in
ethanol, which allows for maintenance of high antigenicity of diverse proteins.
• Because Steedman’s wax is soluble in ethanol, the use of hazardous organic solvents,
often used with other embedding media, can be completely avoided, thus minimizing
health risks and waste disposal costs.
Wax blocks with embedded specimens in the embedding mold (a), and after mounting and trimming
for sectioning (b).
Steedman’s wax
Sectioning and stretching of sections upon a slide. a) The end of the ribbon is being held by a paint
brush during sectioning. b) A drop of water is added to the top of the ribbons on a glass slide, excess
water is then removed from the lower end.
Steedman’s wax
a b
c
d
Immunolocalization of SIMK in root cells of Medicago sativa L. Excised roots were fixed in 3.7 % (w/v)
buffered formaldehyde, dehydrated and embedded in Steedman’s wax. After sectioning and dewaxing, SIMK
was localized by indirect immunofl uorescence with affinity purified SIMK antibody (M23). ( a ) Overview
of SIMK localization in different tissues of root apical meristem. ( b ) SIMK localization in individual root
cells. ( c ) DAPI staining, ( d ) DIC image. Bar = 10 μm
Steedman’s wax
Histological analysis of root meristem cells. The images of paraffin wax sections of maize root meristem
under different stresses are shown. (A) The control group. (B) CuSO4 treatment. (C) Mannitol treatment. (D)
NaCl treatment. (E) Heat treatment. (F) Cold treatment.
Steedman’s wax
Nuclei from seedlings roots under different treatments for 24h were immunostained (H3K9ac, H4K5ac and
H3K4me2 panels) and counterstained with DAPI (DAPI panel). The ‘Merge’ panel shows a merged image of
blue and green staining. More than 200 nuclei were analyzed for every antibody. Bar = 10 mm.
Steedman’s wax
Steedman’s wax
Steedman’s wax
Steedman’s wax
Steedman’s wax
Steedman’s wax
Serial wax sections
Serial wax sections
Placing of serial paraffin sections on microscopy slide
Serial wax sections