The following is a method of our laboratory (improved K3 [Fe (CN) 6] and K4 [Fe (CN) 6] concentration; 90% acetone with ice-pre-cooling reduces endogenous ---) GUS Dyeing analysis refer to SieburtH and Meyerowitz (1997) reports and slightly modified. The specific steps are as follows: Samples are slightly fixed for 90% acetone (in an ice bath) for 10-15 minutes, and then potassium cycyncanide by buffer A (50 mM phosphate buffer, pH 7.2, 5 mM). K3 [Fe (CN) 6], 5 mm ferr cyanide K4 [Fe (CN) 6], 0.5% Triton X-100) washing several times. The sample was then put into GUS dyeing (2 mm x-gluc in buffer A), vacuum 10 minutes (ice bath), and 2 hours at 37 ° C for 2 hours until overnight (depending on the degree of staining of different materials). After dyeing, it was dehydrated by series ethanol (30% ethanol, FAA fixed, 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, 100% ethanol. Each level of ethanol was 10 minutes to 1 hour, FAA fixed for 1 hour). The material after dehydration can be directly transparent to the Hoyer's transparency (Liu and Meinke, 1998). For sections, a part of the material after dehydration is transparent with xylene, paraffin embed (with general paraffin slice treatment step), slice, dewaxing, water (100%, 100%, 95%, 85%, 70%) , 50%, 30%, DDH2O, 2-5 minutes per level), ruthenium red lining, dehydration (30%, 50%, 70%, 85%, 95%, 100%, 100% ethanol, 2- 5 minutes), transparent (1/2 100% ethanol + 1/2 xylene, xylene, xylene, 5 minutes per level), neutral resin seal.
1. Buffer A: 50ml formula final concentration 1M Na2HPO4 1.6 mL 1M kH2PO4 0.9 ml 50 mm Nakpo4 bufferph7.2 0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm 0.1MK4 [Fe (CN) 6] 2.5 mL 5 mm Triton X-100 0.05ml 0.1% Add DDH2O to 50 mL [123 ] 2.Gus dyeing liquid: 50 ml formula final concentration 1M Na2HPO4 1.6 mL 1M KH2PO4 0.9 ml 50MM Nakpo4 bufferph7.2 0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm 0.1MK4 [Fe (CN) 6] 2.5 ml 5 mm Triton X-100 0.05ml 0.1%
X-GLUC 52 mg (400 μL of DMSO) 2MM ADD DDH2O to 50 ml Note: The X-GLUC concentration range is wider, up to 10 mm, usually 2-5MM, most often used 2 mm. General, Concentrations of Ferri / Ferrocyanide Between 0.5 and 5 mm Each Are used for MOST SPECIMENS, 1 mm being a good starting point.
The following is a method of our laboratory (improved K3 [Fe (CN) 6] and K4 [Fe (CN) 6] concentration; 90% acetone with ice-pre-cooling reduces endogenous ---) GUS Dyeing analysis refer to SieburtH and Meyerowitz (1997) reports and slightly modified. The specific steps are as follows: Samples are slightly fixed for 90% acetone (in an ice bath) for 10-15 minutes, and then potassium cycyncanide by buffer A (50 mM phosphate buffer, pH 7.2, 5 mM). K3 [Fe (CN) 6], 5 mm ferr cyanide K4 [Fe (CN) 6], 0.5% Triton X-100) washing several times. The sample was then put into GUS dyeing (2 mm x-gluc in buffer A), vacuum 10 minutes (ice bath), and 2 hours at 37 ° C for 2 hours until overnight (depending on the degree of staining of different materials). After dyeing, it was dehydrated by series ethanol (30% ethanol, FAA fixed, 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, 100% ethanol. Each level of ethanol was 10 minutes to 1 hour, FAA fixed for 1 hour). The material after dehydration can be directly transparent to the Hoyer's transparency (Liu and Meinke, 1998). For sections, a part of the material after dehydration is transparent with xylene, paraffin embed (with general paraffin slice treatment step), slice, dewaxing, water (100%, 100%, 95%, 85%, 70%) , 50%, 30%, DDH2O, 2-5 minutes per level), ruthenium red lining, dehydration (30%, 50%, 70%, 85%, 95%, 100%, 100% ethanol, 2- 5 minutes), transparent (1/2 100% ethanol + 1/2 xylene, xylene, xylene, 5 minutes per level), neutral resin seal.
1. Buffer A: 50ml formula final concentration 1M Na2HPO4 1.6 mL 1M kH2PO4 0.9 ml 50 mm Nakpo4 bufferph7.2 0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm 0.1MK4 [Fe (CN) 6] 2.5 mL 5 mm Triton X-100 0.05ml 0.1% Add DDH2O to 50 mL [123 ] 2.Gus dyeing liquid: 50 ml formula final concentration 1M Na2HPO4 1.6 mL 1M KH2PO4 0.9 ml 50MM Nakpo4 bufferph7.2 0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm 0.1MK4 [Fe (CN) 6] 2.5 ml 5 mm Triton X-100 0.05ml 0.1%
X-GLUC 52 mg (400 μL of DMSO) 2MM ADD DDH2O to 50 ml Note: The X-GLUC concentration range is wider, up to 10 mm, usually 2-5MM, most often used 2 mm. General, Concentrations of Ferri / Ferrocyanide Between 0.5 and 5 mm Each Are used for MOST SPECIMENS, 1 mm being a good starting point.
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GUS Dyeing analysis
refer to SieburtH and Meyerowitz (1997) reports and slightly modified.
The specific steps are as follows: Samples are slightly fixed for 90% acetone (in an ice bath) for 10-15 minutes, and then potassium cycyncanide by buffer A (50 mM phosphate buffer, pH 7.2, 5 mM). K3 [Fe (CN) 6], 5 mm ferr cyanide K4 [Fe (CN) 6], 0.5% Triton X-100) washing several times. The sample was then put into GUS dyeing (2 mm x-gluc in buffer A), vacuum 10 minutes (ice bath), and 2 hours at 37 ° C for 2 hours until overnight (depending on the degree of staining of different materials). After dyeing, it was dehydrated by series ethanol (30% ethanol, FAA fixed, 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, 100% ethanol. Each level of ethanol was 10 minutes to 1 hour, FAA fixed for 1 hour).
The material after dehydration can be directly transparent to the Hoyer's transparency (Liu and Meinke, 1998). For sections, a part of the material after dehydration is transparent with xylene, paraffin embed (with general paraffin slice treatment step), slice, dewaxing, water (100%, 100%, 95%, 85%, 70%) , 50%, 30%, DDH2O, 2-5 minutes per level), ruthenium red lining, dehydration (30%, 50%, 70%, 85%, 95%, 100%, 100% ethanol, 2- 5 minutes), transparent (1/2 100% ethanol + 1/2 xylene, xylene, xylene, 5 minutes per level), neutral resin seal.
1. Buffer A: 50ml formula
final concentration
1M Na2HPO4 1.6 mL
1M kH2PO4 0.9 ml 50 mm Nakpo4 bufferph7.2
0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm
0.1MK4 [Fe (CN) 6] 2.5 mL 5 mm
Triton X-100 0.05ml 0.1%
Add DDH2O to 50 mL [123 ] 2.Gus dyeing liquid: 50 ml formula
final concentration
1M Na2HPO4 1.6 mL
1M KH2PO4 0.9 ml 50MM Nakpo4 bufferph7.2
0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm
0.1MK4 [Fe (CN) 6] 2.5 ml 5 mm
Triton X-100 0.05ml 0.1%
X-GLUC 52 mg (400 μL of DMSO) 2MM
ADD DDH2O to 50 ml
Note: The X-GLUC concentration range is wider, up to 10 mm, usually 2-5MM, most often used 2 mm.
General, Concentrations of Ferri / Ferrocyanide Between 0.5 and 5 mm Each Are used for MOST SPECIMENS, 1 mm being a good starting point.
GUS Dyeing analysis
refer to SieburtH and Meyerowitz (1997) reports and slightly modified.
The specific steps are as follows: Samples are slightly fixed for 90% acetone (in an ice bath) for 10-15 minutes, and then potassium cycyncanide by buffer A (50 mM phosphate buffer, pH 7.2, 5 mM). K3 [Fe (CN) 6], 5 mm ferr cyanide K4 [Fe (CN) 6], 0.5% Triton X-100) washing several times. The sample was then put into GUS dyeing (2 mm x-gluc in buffer A), vacuum 10 minutes (ice bath), and 2 hours at 37 ° C for 2 hours until overnight (depending on the degree of staining of different materials). After dyeing, it was dehydrated by series ethanol (30% ethanol, FAA fixed, 70% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, 100% ethanol. Each level of ethanol was 10 minutes to 1 hour, FAA fixed for 1 hour).
The material after dehydration can be directly transparent to the Hoyer's transparency (Liu and Meinke, 1998). For sections, a part of the material after dehydration is transparent with xylene, paraffin embed (with general paraffin slice treatment step), slice, dewaxing, water (100%, 100%, 95%, 85%, 70%) , 50%, 30%, DDH2O, 2-5 minutes per level), ruthenium red lining, dehydration (30%, 50%, 70%, 85%, 95%, 100%, 100% ethanol, 2- 5 minutes), transparent (1/2 100% ethanol + 1/2 xylene, xylene, xylene, 5 minutes per level), neutral resin seal.
1. Buffer A: 50ml formula
final concentration
1M Na2HPO4 1.6 mL
1M kH2PO4 0.9 ml 50 mm Nakpo4 bufferph7.2
0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm
0.1MK4 [Fe (CN) 6] 2.5 mL 5 mm
Triton X-100 0.05ml 0.1%
Add DDH2O to 50 mL [123 ] 2.Gus dyeing liquid: 50 ml formula
final concentration
1M Na2HPO4 1.6 mL
1M KH2PO4 0.9 ml 50MM Nakpo4 bufferph7.2
0.1MK3 [Fe (CN) 6] 2.5 mL 5 mm
0.1MK4 [Fe (CN) 6] 2.5 ml 5 mm
Triton X-100 0.05ml 0.1%
X-GLUC 52 mg (400 μL of DMSO) 2MM
ADD DDH2O to 50 ml
Note: The X-GLUC concentration range is wider, up to 10 mm, usually 2-5MM, most often used 2 mm.
General, Concentrations of Ferri / Ferrocyanide Between 0.5 and 5 mm Each Are used for MOST SPECIMENS, 1 mm being a good starting point.
More
VOTE