Mitotic metaphase chromosomes were prepared by using the root tips according to the procedure described by She et al. (2006) (link). The actively growing root tips were collected from potted plants and treated with saturated α-bromonaphthalene for 1.5 h at 28 °C, fixed in 3:1 (v/v) methanol/glacial acetic acid for at least 12 h at room temperature, and then stored at 4 °C until use. The fixed root tips were then washed in double distilled water and citrate buffer (0.01 mM citric acid-sodium citrate, pH 4.5) for 10 min each and incubated in a mixture of 2% cellulase R-10 (Yakult Pharmaceutical Industry, Tokyo, Japan), 2% pectolyase Y-23 (Yakult Pharmaceuticals), and 2% macerozyme R-10 (Sigma-Aldrich, Steinhem, Germany) in citric acid buffer at 28 °C for 2.5~3 h. Root tips were transferred to a glass slide along with the fixative and dissected using fine-pointed forceps. Finally, the slides were dried above a flame. Good preparations were selected by Olympus BX60 phase contrast microscope, and then stored at -20 °C.
Macerozyme r 10
Manufactured by Merck Group
Sourced in Germany, United States
Macerozyme R-10 is a commercial enzyme preparation derived from the fungus Rhizopus. It is primarily used for the isolation and purification of plant cells, protoplasts, and other plant-based materials. The enzyme exhibits cellulolytic and pectinolytic activities, which facilitate the breakdown of plant cell walls. Macerozyme R-10 is commonly used in plant tissue culture and biotechnology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Driselase, by Merck Group (2 mentions)
Bx60 phase contrast microscope, by Olympus (1 mentions)
Pectinase, by Merck Group (1 mentions)
Cellulase r10, by Merck Group (1 mentions)
Fumonisin b1, by Merck Group (1 mentions)
Myriocin, by Merck Group (1 mentions)
Cellulase onozuka rs, by Merck Group (1 mentions)
Lyticase, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis kit, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
5 protocols using macerozyme r 10
1
Preparation of Mitotic Chromosomes from Plant Root Tips
2
Protoplast Isolation from Leaf and Root
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The enzyme solution for leaf protoplast isolation contained 0.5 M sucrose for osmotic adjustment, 0.5% Cellulase R10 (C8001, Duchefa Haarlem, the Netherland Company), 0.4% Macerozyme R10 (M8002, Duchefa Haarlem, the Netherland Company), 5 mM MES, and 5 mM Ca(NO3)2 pH 5,4. The modified W5 (W5m) solution [39 (link)] contained 125 mM Ca(NO3)2, 154 mM NaCl, 5 mM KCl, 5 mM glucose, and 2 mM MES. The enzyme solution for root protoplast isolation contained modified W5m salts as osmotic adjustment, 1 g/L sucrose, 0.9% Cellulase R10; 0.4% Macerozyme R10; 0.5% Pectinase (P4716, Sigma-Aldrich St. Louis, Mo., U.S.A), 0.6% Driselase (D9515, St. Louis, Mo., U.S.A), and 5 mM MES, pH 5.4. All enzyme solutions were pre-warmed at 55°C for 10 min to inactivate DNAse/proteases and then filtered through 0.22 µm filters. Sterile enzyme solutions could be stored at 4 °C for up to 1 month without any reduction in activity.
3
Lipid Extraction Reagents Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellulase Onozuka RS, diphenileniodonium (DPI), fumonisin B1 (FB1), macerozymeR-10 and myriocin were obtained from Sigma-Aldrich, (St. Louis, MO, USA). Long-chain bases SN and PS were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). SilwetL-77 was obtained from Chemtura Corporation México, S de RL de CV (Mexico City, Mexico).
4
Annexin V-FITC Assay for Fungal Cell Death
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exposed phosphatidylserine in mASAL treated R. solani hyphae was detected using FITC-conjugated annexin V (Annexin-V FITC Apoptosis Kit, Sigma) as described by Madeo et al. [29 (link)] with some modifications. Both control (treated only with PBS) and mASAL treated (20 μg/ml for 48 h) fungal mycelia were harvested and washed with sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl2, and 35 mM K2HPO4, pH 6.8). The cell walls were digested with 2 % Macerozyme R-10 (Sigma) and 15 U/ml lyticase (Sigma) in sorbitol buffer for approximately 3 h at 28 °C. The cells were harvested and washed with binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) containing 1.2 M Sorbitol (binding-sorbitol buffer). To 96 μl hyphal suspensions in binding-sorbitol buffer, annexin V-FITC and PI are added to a final concentration of 1.2 μg/ml and 5 μg/ml respectively. The resulting suspension was then incubated at room temperature for 20–30 min. Following this the cells were immediately visualized using a confocal laser scanning microscope. A filter for FITC (excitation at 450–500 nm and emission at 515–565 nm) and PI (excitation at 550/25 nm and emission at 605/70 nm) was used. The experiments were performed in triplicate.
5
Immunohistochemical Analysis of Epidermal Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunohistochemical analyses using epidermal fragments were performed as described previously (Hayashi et al., 2011 (link); Ando & Kinoshita, 2018 ). In brief, epidermal fragments were fixed with 4% (w/v) formaldehyde with 0.1% (w/w) glutaraldehyde for 2 h at 4°C. After washing, epidermal fragments were transferred to pure methanol to remove Chl, then washed with Milli‐Q water and mounted on a cover glass coated with 0.1% (w/v) poly‐l ‐lysine (Sigma‐Aldrich). The specimens were digested with 3% (w/v) Driselase (Sigma‐Aldrich) and 0.5% (w/v) Macerozyme R‐10 in PBS for 45 min at 37°C, then permeabilised with 3% (w/w) Triton X‐100 in PBS for 30 min at room temperature. Blocking and antisera application were conducted as described above.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!