Visualisation of pectins and proteins by microscopy … report 87.pdf · Visualisation of pectins...

Visualisation of pectins and proteins by microscopy

Joanna Hornatowska

2005

According to Innventia Confidentiality Policy this report is public since 2011-02-04

Visualisation of pectins and proteins by microscopy

Joanna Hornatowska

STFI-Packforsk report no.: 87 | 2005

Cluster:Mechanical pulp Restricted distribution to: Eka Chemicals, Holmen Paper, Norske Skog, Stora Enso,

Södra Cell, Metsäliitto Group, Myllykoski, UPM-Kymmene

a report from STFI-Packforsk

According to STFI-Packforsk's Confidentiality Policy this report is assigned category 2

Visualisation of pectins och proteins

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Acknowledgements This work was carried out within Mechanical Pulp cluster program 2003-2005 and in cooperation with KCL as a continuation of the KCL-STFI joint research program in the mechanical pulping area, effective up to the end of 2003.




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Table of contents Page

1 Summary..................................................................................................... 4

2 Introduction ................................................................................................ 5

3 Experimental .............................................................................................. 6

3.1 Material.............................................................................................. 6

3.2 Methods............................................................................................. 6 3.2.1 Pectin staining.............................................................................................. 6 3.2.2 Protein staining ............................................................................................ 7 3.2.3 X-ray microanalysis by ESEM-EDS ............................................................. 9

4 Results and discussion ........................................................................... 10

5 Conclusions ............................................................................................. 19

6 References................................................................................................ 20




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1 Summary The purpose of this work was to find and adapt relatively simple and user-friendly microscopic methods for localisation and visualisation of pectins and proteins in wood and on fibre surfaces. The location of these compounds in the fibre cell walls provides valuable complementary information to chemical analysis.

Some histological, well established procedures commonly used in cell biology for location and visualisation of pectins and proteins, were applied and tested on wood samples. Specifically, ruthenium red and hydroxylamine-ferric chloride staining were used for location of unesterified and methylesterified pectins respectively. Tannic acid-ferric chloride staining localizing calcium pectate was also examined. For protein localisation amido black, mercuric bromophenol blue and aniline blue staining procedures were used and tested. All the staining methods are suited for light microscope observation.

Both unesterified and methylesterified pectins were found in the middle lamella, in the primary cell wall/S1, in the bordered pits and in ray cells. The intensity of the stains in the raw wood samples and reference chips were moderate and similar in both cases and did not allow estimation of a dominance of the different pectins.

The effect of chemical treatments used in the simultaneously running chemimechanical project had a visible impact on pectins. The chips treated by 0.05 M and 0.12 M oxalate buffer showed clearly lower intensity of the red stain indicating a decrease of acidic pectins. All the applied treatments, i.e. 0.05 M and 0.12 M oxalate buffer, 0.04 M and 0.08 M urea decreased the intensity of the stain used for methylesterified pectins. This was especially visible in the corners of the middle lamella in latewood areas. The strongest effects were observed on 0.08 M urea treated chips.

Amido black staining for proteins stained the primary cell wall/S1, pit membranes, partly ray cells and occasionally corners of the middle lamella pale blue. It seems that proteins can occur at the same sites as pectin, however only very sporadically in the middle lamella.

Tannic acid-FeCl2, mercuric bromophenol and aniline blue staining gave doubtful results or too weak staining for light microscope observation.




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2 Introduction In the first part of the project some chemical treatments were carried out on wood sticks. An evaluation of crack tendency in the wood sticks by electron microscopy indicated that chemicals affected the outer cell layers (Olsson 2005). A question was raised regarding mechanisms lying behind those reactions and a hypothesis was raised that chemicals react with pectins and/or proteins causing their dissolution and also weaken the bonding between the middle lamella and the primary cell wall. Therefore localisation and visualization of pectins and proteins was a main object of this part of the project.

Pectins are acidic, non-cellulosic polysaccharides ocurring in the cell walls (primary and secondary) and in the cytoplasm. They also are present in the middle lamella and their function there is for adhesion of adjacent cells. The basic component of pectin is D-galactopyranose (galacturonans). Galacturonan was found either methylesterified or unesterified in the cell walls. Westermark and Vennigerholz (1995) reported that lignified wood contained a majority of methylesterified galacturonan. Recent investigations have shown a distribution of homogalacturonans with low and high degree of methyl esterification in cambium and mature wood by immunolabelling with monoclonal antibodies JIM5 and JIM7 (Hafrén 1999, Hafrèn et al. 2000). In unlignified cambium a similar distribution of acidic and esterified pectin was found in middle lamella, ray cell walls and pit membranes. In lignified mature wood both acidic and methylesterified pectin were found in the middle lamella but methylesterified pectin was dominant.

Proteins in plants are macromolecules composed of carbohydrates and protein chain of L-α-amino acids and are generally called glycoproteins. The structural proteins such as extensin, lectins, arabinogalactan-protein and enzymes as peroxidase, phosphatases, glycosyl hudrolases etc. are included into glycoproteins. Still little is known about the structural attachment of proteins, their physiological and biochemical functions. It was reported that the protein fraction in primary cell walls constitutes about 10% of the mass of the primary cell wall (Knox 1990). Regarding the functions of protein, arabinogalactan protein was studied and it was proposed that its function is for binding pectins (Baldwin et al. 1993). Most of the investigations were done on flowering plants, vegetables and fruits and only a few studies have been carried out on wood material.

Some histological, well established procedures used in cell biology for location and visualization of pectins and proteins, have been applied and tested on wood samples (Gurr 1965, Krishnamurthy 1999, Reeve 1959, Sabba and Lulai 2002). Specifically, ruthenium red and hydroxylamine-ferric chloride staining were used for location of unesterified and methylesterified pectins respectively. Tannic acid-ferric chloride staining localizing calcium pectate has been also examined.

For protein localisation amido black, mercuric bromophenol blue and aniline blue staining procedures have been used.




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3 Experimental

3.1 Material Spruce (Picea abies) was used as wood raw material. Steam treated chips of spruce (15 min at 100 ˚C) as a reference, chips treated by oxalate buffer with two concentrations of 0.05 M and 0.12 M and by urea with two concentrations of 0.08 M and 0.04 M were analysed.

Small pieces of samples were sectioned either manually by razor blade or by a sledge microtome. Sections cut by sledge microtome had a thickness about 25 µm. No fixatives were used. All the sections were stored for a short time in deionised water in a refrigerator at about 4 ˚C before staining.

Small sticks of wood and chip samples were also embedded in LR-White methacrylate resin. Sections for light microscopy and confocal laser scanning microscopy were cut 2µm thick by Leica rotation microtome.

3.2 Methods

3.2.1 Pectin staining Three different staining procedures were used to localise and visualise pectins.

Ruthenium red The inorganic dye ruthenium red [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6

. 4H2O has been commonly used as a standard pectin stain in plant tissue and it stains unesterified (acidic) pectins. The staining reaction is not very clear. According to Sterling (1970) staining is effective when the pectin molecule has two negative charges 0.42 nm apart. Methylesterification of the carboxyl groups of adjacent galacturonic acids residues eliminates the negative charges required for binding. Therefore the stain can be used to differentiate between acidic and methylesterified pectins. At low pH, the carboxyl groups are dissociated and the stain may form a salt linkage with anionic groups by electrostatic binding. Staining procedure: 1. Place the material in 0.02 % aqueous ruthenium red solution for 30 min 2. Mount in glycerol 3. Observe under light microscopy The red or pink colour developed by staining indicates presence of unesterified (acidic) pectins.




7 (21) Hydroxylamine-ferric chloride reaction Alkaline hydroxylamine hydrochloride reacts with methyl esters of pectins producing hydroxamic acids. The hydroxamic acid reacts with ferritic ions forming red complexes. The intensity of colour is proportional to the degree of methyl esterification in pectin. Thus, this reaction allows qualitative microscopic localization of the methylesterified pectins. This test is chemically selective and is reckoned as superior to the nonspecific staining obtained with ruthenium red. Staining procedure: 1. Place the material on a slide and cover it by a few drops of fresh mixed alkaline hydroxylamine solution for 5-10 min. The solution contain equal volumes of sodium hydroxide (14 g in 100 ml of 60% ethanol) and hydroxylamine hydrochloride (14 g in 100 ml of 60% ethanol) 2. Add 5 to 10 drops of a solution containing 1 volume concentrated HCl and 2 volumes 95% ethanol. 3. Remove the solution from the material. 4. Add 10% ferric chloride solution in 60% ethanol containing 0.1 N HCL. 5. Dehydrate or clear the material and observe under microscope. When esterified pectins are present a distinct red colour develops. The intensity of the red colour depends on the amount of esterified pectins and degree of esterification.

Tannic acid-ferric chloride This stain is frequently used for studying an occurrence of pectic materials in the primary wall, mostly in plant anatomy. Calcium pectate stains black or blue black. The exact mechanism of the staining reaction is not known. Staining procedure:

1. Place the material in 1% aqueous solution of tannic acid for 10 min. 2. Wash in running water 10 min. 3. Place in 3% aqueos solution of ferric chloride for 3 to 10 min. 4. Wash and observe. If the cell walls are stained black or blue-black, the staining

can be stopped. If not, repeat the complete procedure from the beginning until black or blue-black colour will be developed.

3.2.2 Protein staining Ninhydrine, a commonly used agent in forensic applications visualising proteins and a specific protein stain for fluorescence microscopy SYPRO Ruby protein blot stain were tested. The tests gave negative results and therefore the following staining procedures were chosen to localise non-specific cell wall proteins:




8 (21) Amido Black 10 B In the amido black staining, the proteins become positively charged by acetic acid and react with amido black reagent giving blue colour. Staining procedure:

1. Place sections of fresh or embedded tissue for 30 min in staining (0.5 g amido black, 5 g mercuric chloride, 5 ml glacial acetic acid, 100 ml distilled water)

2. Wash in 2 % acetic acid for 5 min three times

3. Wash in distilled water

4. Mount in glycerine.

Cell wall proteins stain blue.

Mercuric bromophenol blue In the mercuric bromophenol staining, the proteins become positively charged by acetic acid and form electrovalent salt with anionic bromophenol blue through the assistance of mercuric chloride.

Staining procedure:

1. Place sections in a staining mixture 15-30 min (10 g mercuric chloride, 100 mg bromophenol blue and 100 ml distilled water)

2. Wash in 0.5% acetic acid for 1 to 20 min

3. Wash in distilled water for not more than 3 min (otherwise the dye could be removed)

4. Dip in absolute tertiary butanol for 1 min, change used absolute tertiary butanol to a fresh one and place sections again for 2 min

5. Clear and mount in mounting medium DPX.

Proteins bind the stain and give a blue color. The intensity of the blue color depends on the amount of existing proteins.

Aniline blue-black Free amino groups of proteins become positively charged at low pH. Cationic proteins bind with aniline blue-black forming electrovalent salt.

Staining procedure:

1. Place sections in a staining mixture at 50-60 ˚C for 10 min (1 g aniline blue-black in 7% aqueous acetic acid to make a total volume of 100 ml)

2. Immerse sections in 7% aqueous acetic acid

3. Mount in 5% v/v acetic acid in glycerol

Proteins are stained deep blue to bluish black




9 (21) All observations of stained sections were carried out by light microscopy in bright field technique (Zeiss Axioplan) and some of the stained sections were examined by a confocal laser scanning microscope (CLSM, BioRad Radiance 2000). The CLSM is equipped with a Kr/Ar laser (excitation wave length 488 and 568 nm).

3.2.3 X-ray microanalysis by ESEM-EDS X-ray analysis were carried out on the stained sections by Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy (ESEM-EDS, Philips XL 30 ESEM-FEG). The technique is described elsewhere (Newbury et al. 1987).

Only sections stained by inorganical stains forming either ruthenium salts or iron salts were examined by X-ray microanalysis.




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4 Results and discussion

Pectins Three stains were used for localization of pectins in raw wood samples, reference chips and chemically treated chips. The used stains were ruthenium red, hydroxylamine-ferric chloride and tannic acid-ferric chloride.

The raw wood sections and the steam treated reference chip sections were stained very similarly and no differences could be observed in staining sites and color intensity in all staining procedures both for pectins and proteins, see examples in Figure 1.

a) ruthenium red staining of raw wood b) ruthenium red staining of reference chips

c) amido black staining of raw wood d) amido black staining of reference chips

Figure 1. Light microscope micrographs showing staining results of sections; a) and b)staining for pectin; red or pink colours shows sites of pectin c) and d) staining for protein; blue colour shows protein sites. The length of the micrographs is 240 µm.

Ruthenium red stains primarily unesterified pectins. The pectins became red or pink coloured. The higher staining intensity the higher the amount of pectins. The red colour was observed mostly in the middle lamella and on boundaries between the middle lamella and the secondary fibre cell wall (primary cell wall and probably S1).




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a) earlywood of reference chips b) latewood of reference chips

c) earlywood of 0.05 M oxalate treated chips d) latewood of 0.05 Moxalate treated chips

e) earlywood of 0.08 M urea treated chips f) latewood of 0.08 M urea treated chips

g) earlywood of 0.04 M urea treated chips h) latewood of 0.04 M urea treated chips

Figure 2. Micrographs showing sections stained by ruthenium red staining for unesterified pectins. The length of the micrographs is 240 µm.




12 (21) The intensive red colour was also observed in ray cells and pits, especially in bordered pits/pit membrane. As could be seen in the micrographs in Figure 2, the pectins were present in all the samples, however the staining intensity was weaker in samples treated by 0.05 M and 0.12 M oxalate buffer. In Figure 2 shows only sections treated by 0.05 M oxalate buffer due to the almost identical appearance of sections treated by 0.12 M oxalate buffer. The staining intensity of sections treated by urea was very similar to that of the reference chips indicating no special changes in content of acidic pectin. On the other hand, the differences could be very small and not possible to register by eye in a light microscope.

It should be mentioned that colours observed in light microscope directly are considerably better visible than these on micrographs.

a) unstained earlywood b) ruthenium red stained earlywood

c) unstained latewood wood d) ruthenium red stained latewood

Figure 3. CLSM micrographs illustrating presence of lignin in unstained wood sections in a) and c) and presence of lignin and pectins in stained wood sections by ruthenium red b) and d). Observe stronger fluorescence of stained sections. The micrographs size is 196 x 196 µm.

The sections stained by ruthenium red were also examined by CLSM and showed strong fluorescence in the middle lamella, the primary/S1 cell wall and the pit membranes. The




13 (21) control test was carried out on unstained raw wood sections to compare the intensity of lignin auto-fluorescence especially in the middle lamella region. Unfortunately, the strongly stained pectin sites in the middle lamella coincided with the strong lignin auto-fluorescence in the same areas, see micrographs in Figure 3.

Thus CLSM examination of samples stained by ruthenium red failed though a stronger signal from ruthenium red stained samples, compare micrographs in Figure 3.

a) reference chips b) 0.05 M oxalate buffer

c)0.08 M urea d) 0.04 M urea

Figure 4. Light microscope micrographs illustrating results of hydroxylamine-ferric chloride staining. The length of the micrographs is 240 µm.

Hydroxylamine-ferric chloride stains methylesterified pectins. The obtained colours varied from red to pink and pink-orange. The red colour was mostly observed in the primary cell walls/S1 and the pink-orange one in the middle lamella. The intensity of the pink-orange colour was strongest in the wood and reference chips and weaker in oxalate and urea treated chips, especially in sample treated by 0.08 M urea, see and compare micrographs in Figure 4. The decreasing intensity of the stain in the middle lamella indicates decreasing amount of pectins or changes in their degree of methylesterification.




14 (21) Hydroxylamin-FeCl2 staining was also tested on TMP and bleached sulphate fibres. The stain only gave a very weak colour of TMP-fibre surfaces. The bleached sulphate fibres remained unstained, see Figure 5.

a) TMP fibres b) bleached sulphate fibres

Figure 5. Light microscope micrographs illustrating TMP and bleached fibres stained by hydroxylamin-FeCl2 staining. The length of the micrographs is 580 µm.

Additional control staining tests were carried out on wood sections after two chemical treatments dissolving pectins. One treatment was of 0.046 M ammonium oxalate in 90˚C for 12 h and the second one 4% NaOH in room temperature under 4 h. Both ruthenium red and hydroxylamine-ferric chloride stain were used. The staining results are illustrated in Figure 6.

As could be seen in Figure 6 wood sections treated by 0.046 M ammonium oxalate show coloration similar to that achieved by oxalate buffers. While treatment with 4% NaOH gave surprisingly strong red coloration by ruthenium red stain. Such a strong red color was not observed earlier even in raw untreated wood. 4% NaOH solution dissolves not only pectins and proteins but also hemicelluloses. There is no ready explanation for this strong color reaction and what was stained. The presumption is that lignin could be stained by ruthenium red when hemicelluloses are partly dissolved. Therefore the staining results should be treated very carefully. No coloration was obtained by hydroxylamine-ferric chloride stain applied after 4% NaOH treatment.




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a) 0.046 M ammonium oxalate b) 4% NaOH

c) 0.046 M ammonium oxalate d) 4% NaOH

Figure 6. Micrographs illustrating staining results after dissolving pectins from wood-section a) and b) ruthenium red stain, c) and d) hydroxylamine-ferric chloride stain. The length of the micrographs is 240 µm.

Tannic acid-ferric chloride staining, which stains calcium pectate black was also tested. It is believed that unesterified pectins form complexes with calcium ions and therefore this stain was decided to be used to localise sites of calcium pectate. The black colour was observed not only in the middle lamella, primary cell walls, rays and pits membranes but also in the whole fibre walls, especially in latewood in raw wood samples and reference chips. Therefore the staining results were uncertain and should be repeated after a pectin dissolving procedure. However, considerably weaker staining were observed in 0.08 M urea treated chips.

Due to the inorganic origin of ruthenium red and ferritic compounds formed during hydroxylamine-FeCl2 and tannic acid-FeCl2 staining an idea was rised to check the chemical composition of the stained sections and indirectly to determine amounts of pectins in the sections. Therefore X-ray analyses were carried out on the stained sections by ESEM-EDS. The results are presented in Table 1 below:




16 (21) Table 1. Results of semi-quantitative X-ray analysis carried out on stained sections of untreated wood and chips treated by 0.05 M and 0.12 M oxalate buffer and urea of 0.08 and 0.04 M concentration. 0.046 M ammonium oxalate and 4% NaOH wood treatments dissolving pectins are also included.

Sample

Staining

Wood untrea

ted

0.05 M oxalate buffer

0.12 M oxalate buffer

0.04 M urea

0.08 M urea

0.046 M ammo-nium

oxalate

4 % NaOH

Ruthenium red stain, Ru wt %

Earlywood Latewood

1.4 1.0

0.8 0.5

0.9 0.8

1.1 0.7

0.8 0.6

1.0 1.0

1.7 1.2

Hydroxylamin-FeCl2, Fe wt %

Earlywood Latewood

1.6 1.4

1.1 0.8

0.8 1.2

1.3 1.4

1.5 1.2

1.1 1.2

1.3 1.7

Tannic acid- FeCl2, Fe wt %

Earlywood Latewood

0.4 2.2

0.3 1.8

- -

0.2 2.6

0.2 1.2

- -

- -

A lack of a value in the table above means that analyses were not carried out. From the results in the table 1 it might be inferred that oxalate buffer as well as urea treatments had an impact on pectin levels in the chips. Both th amount of ruthenium and iron deacreased somewhat compared to the untreated wood sample. However, these results should be treated very carefully because the X-ray analyses were carried out only on two series of stained sections (five earlywood and five latewood areas in each serie). On the other hand, the X-ray results correspond with the observations by eyes, where weaker staining was observed for oxalate buffers and 0.08 M urea. Stainings performed after dissolution of pectins with 0.046 M ammonium oxalate show the decreased amount of Ru and Fe in earlywood but not in latewood areas. After 4% NaOH treatment the amount of Ru increased. It was also observed in stronger red colour in the wood sections, see Figure 6. Calcium was not detected in any of the sections by this method.

Proteins Three histological protein stains as amido black, mercuric bromophenol blue and aniline blue-black were tested on wood sections due to the negative staining results obtained by ninhydrin and SYPRO Ruby protein blot stain.

Amido black stains cell wall proteins by producing a blue color. The staining coloured the fibre cell walls pale blue and more intensive the primary cell wall/S1. Therefore 2




17 (21) µm sections of the embedded samples were used for this stain to avoid an overstaining of the fibre walls. The primary cell wall/S1 appeared pale blue indicating a moderate occurrence of proteins. The middle lamella was also partially stained. However, no special differences could be observed between the samples after different chemical treatments, se micrographs in Figur 7.

a) raw wood b) oxalate buffer

c) 0.08 urea d) 0.04 urea

Figur 7. Light microscope micrographs illustrating an occurrence of proteins stained by amido black staining. 2 µm sections were stained. The length of the micrographs is 120 µm.

Mercuric bromophenol blue stains a wide variety of reactive protein groups producing a blue color. This blue color was extremely pale and visible under direct observation of the slide only. The sites of protein were almost impossible to detect in light microscope. Therefore this staining procedure was not continued on the remaining samples. This very pale blue colour indicated a small amount of proteins in the fibre cell walls.

Aniline blue-black stains proteins deep blue to blue-black. For sectioned fresh tissue, a section thickness preferably smaller than 10 µm is recommeded. Thus 2 µm embedded sections were used. The stain coloured the fibre walls, the middle lamella and ray cells distinctly deep blue. The primary cell wall/S1 was stained somewhat more intensely but the staining result appeared doubtful. Aniline blue is also used in fluorescence




18 (21) microscopy and the stained sections were examinated by CLSM to see if it is possible to register the deeper blue colour in the primary wall/S1 as a stronger signal. The test failed due to a very even staining of the whole fibre cell wall, as is illustrated in Figur 8. Thus this staining procedure was not continued.

a) light microscope

b) CLSM

Figur 8. Micrographs showing raw wood sections stained by aniline blue-black staining. The length of the light microscope micrograph and CLSM micrograph is 120 µm and 196 µm respectively.

More specific stainings have been developed, characterized and their specifity tested within immunocytochemistry. For pectins the monoclonal antibody JIM5 is recommended due to its specificity against acidic unesterified or very low esterified pectins.This antibody was successfully applied in some studies (Hafrén 1999, Bradley 1988, Van den Bosch et al. 1989). Monoclonal antibody JIM 7 is specific for methylesterified pectins with esterification ranging from 35 to 90 %. For protein localization there are some specific monoclonal and polyclonal antibodies which were also tested and characterized scrupulously.

The largest problem is that antibodies such as JIM5, JIM7 and JIM13 (the last specific for protein) are not commercially available. The production of antibodies demands much more knowledge and experience in biochemistry.




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5 Conclusions Rhutenium red and hydroxylamine-ferric chloride stained unesterified (acidic) and methylesterified pectin respectively. In the raw wood and the reference chips, the pectin sites were localised to middle lamella, primary fiber wall/S1, ray cells and pits/pit membranes. The intensity of the stained pectine sites was moderate and similar in both cases. Thus this did not allow for an estimation of the dominance of one or the other of the pectins.

Chemical treatments made by 0.05 M and 0.12 M oxalate buffer and urea with two concentration 0.04 M and 0.08 M had a visible impact on the pectins in the wood. The chips treated by oxalate buffers showed clearly lower intensity of the red stain indicating decrease of acidic pectins. All the applied treatments, i.e. oxalate buffers and urea with two concentrations decreased the intensity of the stain used for methylesterified pectins. This was especially visible in the corners of the middle lamella in latewood areas. The strongest effects were observed on 0.08 M urea treated chips.

Amido black staining for proteins stained the primary cell wall/S1, pit membranes, partly ray cells and occasionally corners of the middle lamella pale blue. It seems that proteins can occur at the same sites as pectin, however only sporadically in the middle lamella. This should be checked by more advanced and sensitive microscopic methods.

Tannic acid-FeCl2, aniline blue-black and mercuric bromophenol staining gave either doubtful results or too weak staining for light microscope observation.

Immunocytochemistry can be used for more specific and more exact location of pectins and proteins. Immunolabelling require monoclonal or polyclonal antibodies which are not always available commercially. This kind of studies can be performed in cooperation with universities or companies developing/producing antibodies.




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6 References Baldwin T, McCann M C and Roberts K A A novel hydroxyproline-deficient arabinogalactan protein secreted by suspension-cultured cells of Daucus carota; Plant Physiol. 103, 115 (1993)

Bradley D J, Wood E A, Larkins A P, Galfie G, Butcher G W and Brewin N J Isolation of monoclonal antibodies reacting with peribacterioid membrance and other components of pea root nodules containing Rhizobium leguminosarum Planta 173, 149 (1988)

Gurr E The rational use of dyes in biology and general staining methods Leonard Hill, London (1965)

Hafrén J Ultrastructure of the wood cell Ph.D. Thesis of Royal Institute of Technology (1999)

Hafrén J, Daniel G and Westermark U The distribution of acidic and esterified pectin in cambium, developing xylem and mature xylem of Pinus Sylvestris, IAWA Journal, Vol. 21 (2), 157-168 (2000)

Knox J P Emerging patterns of organization at the plant cell surface J. Cell Sci. 96, 557 (1990)

Krishnamurthy K V Methods in cell wall cytochemistry CRC Press LLC (1999)

Newbury D E, Joy D C, Echlin P, Fiori C E and Goldstein J I Advanced Scanning Electron Microscopy and X-ray Microanalysis Plenum Press, New York (1987)

Olsson A-M Chemimechanical treatments, Part one: Chemical treatments on small wood pieces STFI-Packforsk Report No 6 (2005)

Reeve R M A specific hydroxylamine-ferric chloride reaction for histochemical localization of pectin Stain Technology 34, 209-211 (1959)

Sabba R, Lulai E Histological analysis of the maturation of native and wound periderm in potato (Solanum tuberosum L.) tuber, Annals of Botany 90, 1-10 (2002)

Sterling C Crystal structure of ruthenium redand stereochemistry of its pectin stain Am.J.Bot. 57, 172 (1970)




21 (21) Van der Bosch K A, Bradley D J, Knox J P, Perotto S, Butcher G W and Brewin N Common components of the infection thread matrix and the intracellular space identified by immunocytochemical analysis of pea nodules and uninfected roots EMBO Journal, 8, 335 (1989)

Westermark U and Vennigerholz F Morphological distribution of acidic and methylesterified pectin in the wood cell wall Proc. 8th Intern. Symp. on Wood and Pulping Chemistry 1, 101-106 (1995)


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