can anyone please share with me the protocols or procedures and necessary precautions to be taken while preparing the stains and fixatives for studying the microsporogenesis and pollen development?
Hi dear Surendra,
There are several fixative and staining techniques. I often use FAA (1 ml acetic acid+2 ml formaldehyde+ 17 ml ethanol) for fixation and hematoxylin and eosin staining protocol for staining the tissues. I keep the flowers (depend on the size of them) in fixative for 8-24 h and wash them with running water for several hours. Then, I transfer them to ethanol 50 for 15-30 min and keep them in ethanol 70 until use them for sectioning.
dehydration for embedding
protocol 1
Ethanol (E) 90: 15-30 min
E100: 15-30 min (twice)
3/4 E + 1/4 toluene or Xylene (is better for plant tissues): 15-30 min
2/4E+2/4X : 15-30 min
3/4E+1/4X: 15-30 min
Pure X: 15-30 min (twice)
3/4X+1/4 Paraffin: 15-30 min (in oven 60 Centigrade)
2/4X+2/4P: 15-30 min (in oven 60 Centigrade)
1/4X+3/4P:15-30 min (in oven 60 Centigrade)
P1: 15-30 min
P2: 45 min-1 h
P3: 2 day-2 weeks
protocol 2:
Ethanol (E) 90: 1 h
E100: 1 h (twice)
E+X (1:1): 1 h-1day (depend on amount of lignification and secondary metabolites, such as tannin)
X1: 1 h
X2: 1 h
X3: 1 h-1 day (depend on amount of lignification and secondary metabolites, such as tannin)
P1: 3-4 h
P2: 3-4 h
P3: 3 h- 2 weeks
Try one of these protocol for your samples to find witch of them is better.
You can also read this book: Botanical Microtechnique, John E. Sass
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