Overnight EU-labeled root tips were excised and fixed in freshly made ice-cold ethanol:acetic acid (3:1) fixative for 24 h. The fixative was exchanged once during this time. Roots were then washed 1 × 5 min in distilled water, 2 × 5 min in 10 mM citrate buffer (4 mM citric acid and 6 mM sodium citrate, pH 4.5), and digested by a mixture of cellulase (Onozuka R10, Serva 16419.03), pectolyase (Duchefa, P8004.0001), and cytohelicase (Sigma, C8274), 0.3% (w/v) each in 10 mM citrate buffer, for 25 min at 37°C. Digested root tips were washed once in citrate buffer and transferred to slides. After complete removal of the citrate buffer, root tips were squashed in a drop of 50% acetic acid. Cover slips were removed in liquid nitrogen, and slides were re-fixed in fresh ethanol:acetic acid fixative and air dried. The click iT reaction to detect EU by fluorescence was performed as described above, 200 μl of click iT mixture was applied on each slide. Slides were then washed 3 × 5 min in 1× PBS and stained with DAPI in Vectashield (1 μg ml-1, Vector Laboratories, H100).
Pectolyase
Manufactured by Duchefa Biochemie
Pectolyase is a laboratory enzyme used for the enzymatic digestion of plant cell walls. It acts by breaking down pectin, a major structural component of plant cell walls. Pectolyase is commonly used in plant cell and protoplast isolation procedures.
Automatically generated - may contain errors
Lab products found in correlation
Cytohelicase, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Pectinase, by Merck Group (2 mentions)
Cellulase, by Merck Group (1 mentions)
Hemicellulase, by Merck Group (1 mentions)
Driselase, by Merck Group (1 mentions)
Cellulase r 10, by Duchefa Biochemie (1 mentions)
Macerozyme r10, by Duchefa Biochemie (1 mentions)
Cellulase rs, by Duchefa Biochemie (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Dm5000b microscope, by Leica (1 mentions)
Cellulase, by Duchefa Biochemie (1 mentions)
4 protocols using pectolyase
1
EU-Labeling of Plant Root Tips
2
Optimizing Cell Wall Enzymatic Digestion
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We tested several cell wall digestion enzymes from multiple manufacturers and found the performance of the enzymes varied between manufacturers, as well as between batches from the same manufacturers. We tested driselase (Sigma), cellulase (Sigma), cellulase R10 (Duchefa, Yakult), cellulase RS (Duchefa, Yakult), pectolyase (Duchefa; discontinued), pectinase (Sigma), macerozyme R10 (Duchefa) and hemicellulase (Sigma) for conventional whole-mount protocol. We chose cellulase RS (Duchefa), hemicellulase (Sigma) and pectinase (Sigma) or macerozyme R10 (Duchefa) for expansion microscopy based on their performance and availability.
3
Arabidopsis Floral Bud Chromosome Staining
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Arabidopsis immature floral buds were collected, fixed, excised and processed according to Mokros et al. (2006 (link)). We used mix of three enzymes (0.3% each), in 10 mm citrate buffer – cellulase (Onozuka R10, Serva), pectolyase (Duchefa) and cytohelicase (Sigma, https://www.sigmaaldrich.com/ ). Slides were then stained with DAPI (1 μg ml−1) in Vectashield (Vector Laboratories) and anaphases were count on each slide using Zeiss Axioimager Z1 with specific filter corresponding to DAPI excitation and emission spectra (AHF Analysentechnik, http://www.ahf.de/ ).
4
Chromosome Counting in Riccia fluitans
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Chromosome counting was carried out according to Schwarzacher [53 (link)]. Briefly, thallus tips from freshly started R. fluitans 001TC and R. fluitans BoGa cultures ensuring high cell division rates, were collected and transferred to ice water for 24 h and afterwards incubated in saturated 8-hydroxyquinoline solution for 3 h. Thallus pieces were then fixed overnight in Carnoy’s solution. The next day, thalli were washed five times with 10 mM citric acid buffer (pH 4.6) to remove the fixative. Thalli were then digested with 2.5% pectolyase (Duchefa, Haarlem, The Netherlands), 2.5% pectinase (Sigma-Aldrich, Darmstadt, Germany), and 2.5% cellulase (Duchefa, Haarlem, The Netherlands) for 30 min and washed with ddH2O. DAPI (10 µg/mL) staining was performed on object slides, tissue was then squashed with a cover slip, and staining results were immediately observed using a Leica DM5000 B microscope.
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