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