Two flowers per day from anthesis, two and three days after pollination were fixed in 4% formaldehyde freshly prepared from paraformaldehyde in 1x phosphate saline buffer (PBS) pH7.3, left overnight at 4ºC, and conserved then at 0.1% formaldehyde solution [83 (link)]. Then the pistils were dehydrated in an acetone series (30%, 50%, 70%, 90%, 100%), and embedded in Technovit 8100 (Kulzer and Co, Germany) for two days. The resin was polymerized at 4ºC, and sectioned at 4 μm thickness. Sections were placed in a drop of water on a slide covered with 2% (3-Aminopropyl) triethoxysilane - APTEX (Sigma-Aldrich), and dried at room temperature. Callose was identified with the anticallose antibody (AntiCal) that recognises linear β-(1,3)-glucan segments (anti-β-(1,3)-glucan; immunoglobulin G1), Biosupplies, Australia [49 (link)]. As a secondary antibody, Alexa 488 fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG was used (F-1763; Sigma). Additionally, a monoclonal antibody (mAbs) JIM13 [84 (link)] against AGPs glycosyl epitopes, and one mAb JIM11 [85 (link)] against extensin epitopes were obtained from Carbosource Services (University of Georgia, USA). Secondary antibodies were anti-rat IgG conjugated with the same Alexa 488 used above. Sections were incubated for 5 min in PBS pH7.3 followed by 5% bovine serum albumin (BSA) in PBS for 5 min. Then, sections were incubated at room temperature for 1h with AntiCal primary mAb, JIM13, and JIM11. After that, three washes in PBS of 5 minutes each preceded the incubation for 45 min in the dark with a 1/25 diluted secondary fluorescein isothiocyanate (FITC) conjugated with the antibody in 1% BSA in PBS, followed by three washes in PBS [83 (link)]. Sections were counterstained with calcofluor white for cellulose [86 (link)], mounted in PBS or Mowiol, and examined under a LEICA DM2500 epifluorescence microscope connected to a LEICA DFC320 camera. Filters were 355/455 nm for calcofluor white and 470/525 nm for the Alexa 488 fluorescein label of the antibodies (White Level?=?255; Black Level = 0; ϒ?=?1). Exposur (Exp) times were adapted to the best compromise in overlapping photographs for each antibody: AntiCal, Exp.?=?15.30ms (Calcofluor Exp. = 1.20ms); JIM13 Exp.?=?2.52ms (Calcofluor?=?0.41ms); JIM11, Exp. = 31.59 ms (Calcofluor Exp. = 1.40ms). Brightness and contrasts were adjusted to obtain the sharpest images with the Leica Application Suite software.
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Callose
Callose
Callose is a major cell wall component found in plants that plays a crucial role in various biological processes.
It is a β-1,3-glucan polymer that accumulates in response to biotic and abiotic stresses, contributing to plant defense mechanisms.
Callose deposition is observed in cell plates during cytokinesis, as well as in plasmodesmata, sieve plates, and other specialized plant structures.
Studying the regulation and dynamics of callose is important for understanding plant growth, development, and adaptability to environmental challenges.
PubCompare.ai's AI-driven platform can enhance the reproducibility and accuracy of callose research by helping researchers locate the best protocols from literature, pre-prints, and patents through AI-driven comparisons.
This user-freindly interface and cutting-edge technology can optimize callose experiments and unlock the power of this essential plant biomolecule.
It is a β-1,3-glucan polymer that accumulates in response to biotic and abiotic stresses, contributing to plant defense mechanisms.
Callose deposition is observed in cell plates during cytokinesis, as well as in plasmodesmata, sieve plates, and other specialized plant structures.
Studying the regulation and dynamics of callose is important for understanding plant growth, development, and adaptability to environmental challenges.
PubCompare.ai's AI-driven platform can enhance the reproducibility and accuracy of callose research by helping researchers locate the best protocols from literature, pre-prints, and patents through AI-driven comparisons.
This user-freindly interface and cutting-edge technology can optimize callose experiments and unlock the power of this essential plant biomolecule.
Most cited protocols related to «Callose»
3-(triethoxysilyl)propylamine
Acetone
anti-IgG
Antibodies
Bos taurus
Buffers
calcofluor white
callose
Cellulose
Contrast Media
Epitopes
Flowers
Fluorescein
Formaldehyde
Formalin
Glucans
Immunoglobulins
isothiocyanate
Mice, House
Microscopy
Orosomucoid
paraform
Phosphates
Pistil
Pollination
Resins, Plant
Saline Solution
Serum Albumin
Serum Albumin, Bovine
Anabolism
aniline blue
Bacteria
Biological Assay
callose
DNA, Complementary
Gene Chips
Hypersensitivity
Infection
Luciferases
Magnesium Chloride
Microarray Analysis
Phenotype
Plant Roots
Plants
Promega
Proteins
Pseudomonas Infections
Pseudomonas syringae
Spores
SYBR Green I
Trypan Blue
Ultraviolet Rays
Vaccination
For spray inoculation, conidial suspension (10 ml) containing Tween 20 (250 ppm) and conidia harvested from 12-day-old cultures on OMA plate (1–5×105 conidia/ml) was sprayed onto four-weeks old susceptible rice seedlings (Oryza sativa cv. Nakdongbyeo). Inoculated plants were placed in a dew chamber at 25°C for 24 hours in the dark, and then transferred back to the growth chamber with a photoperiod of 16 hours using fluorescent lights [74] . Disease severity was assessed at seven days after inoculation. The %DLA was recorded to permit more accurate evaluation of the virulence of the mutants. Photographs of diseased rice leaves including eight centimeter long leaf blades were taken. The number of pixels under lesion areas and healthy areas of diseased leaves was calculated by Axiovision image analyzer with the photographs. For microscopic observation of penetration and infectious growth on rice tissue, excised rice leaf sheath of Nakdongbyeo were prepared as previously described [30] (link),[42] and inoculated by conidia suspension (1×104 conidia/ml) on the adaxial surface. After 24, 48 and 96 hours incubation in a moistened box, the sheaths were trimmed to remove chlorophyll enriched plant parts. Remaining epidermal layer of mid vein (three to four cell layers thick) were utilized for microscopic observations. Inoculation on onion epidermis was performed as previously described [75] (link). Fixation and aniline blue staining of rice sheath and onion epidermis were performed as previously described [75] (link). Samples were incubated in lactophenol at room temperature for 1hour and directly mounted with 70% glycerin or transferred into 0.01% aniline blue for 1hour and destained with lactophenol. For 3, 3′-diaminobenzidine (DAB, Sigma, D-8001) staining, samples were incubated in 1mg/ml DAB solution (pH 3.8) at room temperature for 8 hours and destained with clearing solution (ethanol∶acetic acid = 94∶4, v/v) for 1 hour. For observation and scoring penetration rate and IH development, conidia suspension were dropped on onion epidermis and incubated for 72 hours in moistened culture plate. Samples were fixed and stained as rice sheath described above. Extensive IH from single appressoria with no (or scatterd) callose were scored as normal IH, relative short and attenuated IH with accumulated callose were scored as retarded IH, appressorium developing very short IH or penetration peg with strong callose were scored as blocked IH, and appressorium without IH and callose deposition were scored no penetration.
Acetic Acid
Allium cepa
aniline blue
callose
Cells
Chlorophyll
Conidia
Epidermis
Ethanol
Glycerin
Infection
Light
Microscopy
Oryza sativa
Plant Leaves
Plants
Seedlings
Tissues
Tween 20
Vaccination
Veins
Virulence
To monitor pollen tube growth rate in the stigma and the style, following hand pollinations, five flowers -30 gynoecia- were sampled at intervals of two hours up to twelve hours, and later the same number of flowers was daily sampled and fixed in FAA (formalin: acetic acid: 70% ethanol) (1:1:18) [75 ] for seven days after pollination. After being fixed, the pistils were washed three times in distilled water for one hour each, and left in 5% sodium sulphite for 24 h. Samples were autoclaved for 10 min at 1 Kg cm-2 in 5% sodium sulphite, and then the individual styles were squashed onto glass slides with 0.1% aniline blue in 0.1NK3PO4 to visualize callose [76 ,77 (link)] and pollen tubes [41 ]. Pollen tubes were visualized with a LEICA DM2500 fluorescence microscope bearing a 340/400 nm filter, and pollen tube number was counted at the hypanthium entrance. Percentages of styles with pollen tubes at the stylar base were recorded in pollinated pistils fixed at different days after pollination, and were sequentially compared by days after pollination using chi-square homogeneity test at a P?≤?0.05. Both percentages of styles traversed and mean number of pollen tubes at the stylar base were subjected to mean comparison by one way ANOVA, and significant independent groups were separated by Duncan multiple range test at a P?≤?0.05. Statistical analysis was performed with the SPSS software (SPSS Inc., Chicago, USA).
Acetic Acid
aniline blue
callose
Ethanol
Flowers
Formalin
Microscopy, Fluorescence
neuro-oncological ventral antigen 2, human
Pistil
Pollen Tube
Pollination
sodium sulfite
Publicly available sequences of transcripts from GenBank® and DFCI Grape Gene Index databases were analyzed in the set-up of quantitative real-time PCR reactions (qRT-PCR), with SYBRGreen I and TaqMan® chemistry for 15 target genes, selected from the list of differentially expressed genes obtained from the microarray data analysis: vacuolar acid invertase 2 (VvInv2), ADP-glucose pyrophosphorylase (VvAgpL), apocytochrome f precursor (VvAcyt), ethylene receptor (VvEtr), flavanone 3-hydroxylase (VvF3h), histidine-containing phosphotransfer protein involved in cytokinine signal transduction (VvHP), lipoxygenase (VvLox), osmotin-like protein (VvOlp), S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (VvSamt), WRKY54 (VvWrky), callose synthase (VvCaSy), cytokinin oxidase (VvCko), β-1,3-glucanase I (VvGlc1), β-1,3-glucanase II (VvGlc2) and β-1,3-glucanase III (VvGlc3). Primer Express© software (Applied Biosystems) was used for the design of primer pairs. Specificities of the designed amplicons were tested in silico using BLASTn search of public databases [53 (link)]. Primer pairs were designed in the region of microarray oligo design to ensure the compatibility of results between both platforms. Details of the primer design are described in [Additional file 8 ].
Two amplicons published by [15 (link)], i.e. sucrose synthase (VvSuSy) and alcohol dehydrogenase I (VvAdh1)) were added to the set of 15 newly designed amplicons for sample screening. All qRT-PCR reactions were performed on an ABI PRISM® 7900 HT Sequence Detection System (Applied Biosystems) as described in [13 (link)].
The relative quantification approach was used essentially as described in [54 (link)] and [13 (link)] with cytochrome oxidase (Cox; [55 (link)]) and 18S (Eukaryotic 18S rRNA TaqMan endogenous control, Applied Biosystems) as endogenous controls required for the normalization process. Due to low expression values of genes VvSamt and Vvinv2 in samples from healthy leaves (Ct values were above 34 or even undetermined in the higher of the two dilutions used in qRT-PCR reactions). Therefore we replaced the Ct values in all healthy samples that had Ct values above 34.0 with values of 34.0 for the higher and 30.7 for the lower dilution in order for the samples to pass the quality control and to be included into the t-statistics. This procedure is common in the pre-processing of microarray data where negative signal values after background correction (low signal intensity spots) are corrected with an arbitrary offset [56 (link)]. In this way, genes with low expression values in a certain state (in our example in healthy samples) are not excluded from analysis since they may be highly expressed in another state and may be therefore differentially expressed.
The Welch two sample t-test was used to determine statistically significant differences between relative expression ratios of infected and healthy samples with a P = 0.05 as the limit for statistical significance.
Two amplicons published by [15 (link)], i.e. sucrose synthase (VvSuSy) and alcohol dehydrogenase I (VvAdh1)) were added to the set of 15 newly designed amplicons for sample screening. All qRT-PCR reactions were performed on an ABI PRISM® 7900 HT Sequence Detection System (Applied Biosystems) as described in [13 (link)].
The relative quantification approach was used essentially as described in [54 (link)] and [13 (link)] with cytochrome oxidase (Cox; [55 (link)]) and 18S (Eukaryotic 18S rRNA TaqMan endogenous control, Applied Biosystems) as endogenous controls required for the normalization process. Due to low expression values of genes VvSamt and Vvinv2 in samples from healthy leaves (Ct values were above 34 or even undetermined in the higher of the two dilutions used in qRT-PCR reactions). Therefore we replaced the Ct values in all healthy samples that had Ct values above 34.0 with values of 34.0 for the higher and 30.7 for the lower dilution in order for the samples to pass the quality control and to be included into the t-statistics. This procedure is common in the pre-processing of microarray data where negative signal values after background correction (low signal intensity spots) are corrected with an arbitrary offset [56 (link)]. In this way, genes with low expression values in a certain state (in our example in healthy samples) are not excluded from analysis since they may be highly expressed in another state and may be therefore differentially expressed.
The Welch two sample t-test was used to determine statistically significant differences between relative expression ratios of infected and healthy samples with a P = 0.05 as the limit for statistical significance.
Most recents protocols related to «Callose»
The time required to carry out the extraction step is 7 days for chlorophyll clearing and 5 h for effective extraction of the callose. Figure 1 reports in detail the procedure to extract and stain the callose.
Harvest the tissue and freeze it immediately in liquid nitrogen. Grind the samples with a mortar and pestle using liquid nitrogen then weigh (20 mg) and transfer to 2.0 mL screw-cap tubes suitable for use with TissueLyser (cat 85300, Qiagen, Hilden, Germany). Grind the sample a second time with steel beads using TissueLyser (30 s up to 8 times). Keep the powder frozen. Add 1.0 mL of 100% EtOH to the sample. To avoid the formation of clumps, quickly mix the samples (15-20 sec) with TissueLyser. Remove the beads. Incubate the samples at RT and remove the chlorophyll by incubating the samples for seven days on an orbital shaker with several changes of ethanol until all chlorophyll is gone.
After 7 days of washing, centrifuge the samples for 5 min at 10000g, and discard the EtOH. Soak the tissue for 5 min in 1.2 mL ethanol at 50°C in a heat block and mix. Centrifuge for 5 min at 10000g at room temperature. Discard the EtOH. After this step, wash the pellet in 600 mL ethanol and vortex, and centrifuge for 5 min at 4000g at room temperature; repeat this step twice. Dissolve the sample in 1650 mL of 1M NaOH. Sonicate for 20 min and mix regularly to allow the disruption of the cells. Incubate the samples for 90 min at 85°C in a heat block and shake frequently (every 2-3 min). Cool down to room temperature (~10 min), and centrifuge for 10 min at 10000g. Transfer the supernatant to a fresh snap-capped tube. The supernatant can be stored at 4°C for up to 6 months.
For the quantitative determination of extracted callose, supplement 25-100 mL of supernatant with 200-275 mL of NaOH User-friendly protocol for callose extraction. The picture reports step-by-step the protocol followed for the extraction and the quantification of the callose. Before adding the EtOH all the steps should be carried out in frozen conditions. After the addition of aniline blue, the samples may be kept under the foil, to prevent the degradation of the fluorescence. The protocol can be stopped after the extraction of the callose and before the staining. Picture realized with Biorender.
Harvest the tissue and freeze it immediately in liquid nitrogen. Grind the samples with a mortar and pestle using liquid nitrogen then weigh (20 mg) and transfer to 2.0 mL screw-cap tubes suitable for use with TissueLyser (cat 85300, Qiagen, Hilden, Germany). Grind the sample a second time with steel beads using TissueLyser (30 s up to 8 times). Keep the powder frozen. Add 1.0 mL of 100% EtOH to the sample. To avoid the formation of clumps, quickly mix the samples (15-20 sec) with TissueLyser. Remove the beads. Incubate the samples at RT and remove the chlorophyll by incubating the samples for seven days on an orbital shaker with several changes of ethanol until all chlorophyll is gone.
After 7 days of washing, centrifuge the samples for 5 min at 10000g, and discard the EtOH. Soak the tissue for 5 min in 1.2 mL ethanol at 50°C in a heat block and mix. Centrifuge for 5 min at 10000g at room temperature. Discard the EtOH. After this step, wash the pellet in 600 mL ethanol and vortex, and centrifuge for 5 min at 4000g at room temperature; repeat this step twice. Dissolve the sample in 1650 mL of 1M NaOH. Sonicate for 20 min and mix regularly to allow the disruption of the cells. Incubate the samples for 90 min at 85°C in a heat block and shake frequently (every 2-3 min). Cool down to room temperature (~10 min), and centrifuge for 10 min at 10000g. Transfer the supernatant to a fresh snap-capped tube. The supernatant can be stored at 4°C for up to 6 months.
For the quantitative determination of extracted callose, supplement 25-100 mL of supernatant with 200-275 mL of NaOH User-friendly protocol for callose extraction. The picture reports step-by-step the protocol followed for the extraction and the quantification of the callose. Before adding the EtOH all the steps should be carried out in frozen conditions. After the addition of aniline blue, the samples may be kept under the foil, to prevent the degradation of the fluorescence. The protocol can be stopped after the extraction of the callose and before the staining. Picture realized with Biorender.
Callose staining was determined as described previously (Su et al. 2023 (link)). After treating leaves infected with TRV and TRV-RcMYB8 using NaCl solution (0 and 200 mM), they were subjected to a 1-h aniline blue solution (Solarbio, Beijing, China) staining devoid of light. Green fluorescent samples were deemed as callose (Luna et al. 2011 (link)), which were surveilled using an Axio Scope A1 microscope (Carl Zeiss). With the aid of ImageJ (https://imagej.nih·gov/ij/ ), the callose quantity was identified.
Aniline blue staining was used to determine callose deposition level as described by Ton and Mauch-Mani (2004) (link). Leaves were kept overnight in 95% ethanol to remove the chlorophyll, stained in 0.05% aniline blue (in 0.1 M Na 2 HPO 4 phosphate buffer, pH 8), and kept overnight before being mounted on microscope slides. Pictures were taken using a fluorescence microscope (Olympus-IX71, Olympus CO., Japan). Accumulation of callose was quantified using GIMP software.
Callose deposit quantification assays were conducted and images acquired as previously described in Jayaraman et al. (2023) (link). Images were analysed using ImageJ software by determining the average area of a single callose deposit and then callose counts calculated based on total callose deposit area in each image.
Callose staining was carried out as described (66) . Briefly, individual leaf disks were soaked with 0,1% aniline blue solution (in 50 mM potassium phosphate buffer, pH 8.0) containing either 1 μM flg22 (MedChemExpress, NJ, USA), 30 ng/ul dsPVY or control extracts. Leaf disks were further evacuated for 1-2 minutes (< 0.8 Pa) in a vacuum desiccator. Aniline blue fluorescence was imaged 30 minutes after dsPVY/flg22 or control treatment using a Leica TCS SP8 STED 3X confocal microscope (Wetzlar, Germany) with Leica Application Suite X software and using a 405 nm diode laser for preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted February 7, 2024. ; https://doi.org/10.1101/2024.02.07.579064 doi: bioRxiv preprint excitation and filtering the emission at 430-490 nm. 8-bit images were acquired with a HC PL APO 40x/1.30 Oil CS2 objective. Callose fluorescence intensity was quantified with ImageJ software (http://rsbweb.nih. gov/ij/) using the plug-in calloseQuant (66) .
Callose spots were measured in 5-6 images taken from three leaf discs per plant from two different plants for each treatment. Regions of interest selected by calloseQuant were verified visually before measurement. ROIs that were not overlapping with the cell wall or did not contain clear signal above the background were deleted. Individual fluorescence intensities that occurred as outliers from the general distribution of fluorescence intensities (<1%) in the sample were excluded from analysis.
The copyright holder for this this version posted February 7, 2024. ; https://doi.org/10.1101/2024.02.07.579064 doi: bioRxiv preprint excitation and filtering the emission at 430-490 nm. 8-bit images were acquired with a HC PL APO 40x/1.30 Oil CS2 objective. Callose fluorescence intensity was quantified with ImageJ software (http://rsbweb.nih. gov/ij/) using the plug-in calloseQuant (66) .
Callose spots were measured in 5-6 images taken from three leaf discs per plant from two different plants for each treatment. Regions of interest selected by calloseQuant were verified visually before measurement. ROIs that were not overlapping with the cell wall or did not contain clear signal above the background were deleted. Individual fluorescence intensities that occurred as outliers from the general distribution of fluorescence intensities (<1%) in the sample were excluded from analysis.
Top products related to «Callose»
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Aniline blue is a synthetic dye used as a staining agent in various laboratory applications. It is a deep blue or purple colored powder that is soluble in water. Aniline blue is commonly used to stain nucleic acids, carbohydrates, and other biological structures for visualization and analysis purposes.
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The BX51 microscope is an optical microscope designed for a variety of laboratory applications. It features a modular design and offers various illumination and observation methods to accommodate different sample types and research needs.
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The LSM 880 is a laser scanning confocal microscope designed by Zeiss. It is a versatile instrument that provides high-resolution imaging capabilities for a wide range of applications in life science research.
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The Eclipse 80i is a microscope designed for laboratory use. It features an infinity-corrected optical system and offers a range of illumination options. The Eclipse 80i is capable of various imaging techniques, including phase contrast and brightfield microscopy.
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The Leica TCS SP8 is a confocal laser scanning microscope designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research needs. The TCS SP8 provides high-resolution, multi-dimensional imaging capabilities, enabling users to capture detailed, real-time observations of biological samples.
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The Leica DM2500 is a high-performance microscope designed for laboratory applications. It offers a range of features and capabilities to support various research and analysis tasks.
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K2HPO4 is a chemical compound commonly used in laboratory settings. It is a white crystalline powder with the formula K2HPO4. K2HPO4 is a salt of potassium and phosphate and serves as a buffer solution in various laboratory applications.
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TRIzol reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA, DNA, and proteins from a variety of biological samples. The reagent maintains the integrity of the RNA while disrupting cells and dissolving cell components.
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Calcofluor white is a fluorescent brightening agent used in microscopy and staining applications. It binds to cellulose and chitin in cell walls, allowing for the visualization of fungal and other microbial structures.
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3,3'-diaminobenzidine is a chemical compound commonly used as a chromogenic substrate in various laboratory techniques, such as immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It is a sensitive and specific reagent that can be used to detect the presence of target proteins or enzymes in biological samples.
More about "Callose"
Callose, β-1,3-glucan, plant cell wall, biotic stress, abiotic stress, plant defense, cytokinesis, plasmodesmata, sieve plates, plant growth, plant development, plant adaptability, PubCompare.ai, AI-driven platform, reproducibility, accuracy, protocols, literature, pre-prints, patents, AI-driven comparisons, aniline blue, BX51 microscope, LSM 880, Eclipse 80i, TCS SP8, DM2500, K2HPO4, TRIzol reagent, Calcofluor white