We have tried dipping roots for 10 min at PBS for three times. But we found that the roots were becoming translucent but they are still fluorescence. So seeking information regarding the time and frequency of wash with PBS.
@ Anju, You could go up to an hour and then wash them off extensively with PBS buffer before cryo-sectioning. Hope it helps.
Dear Anju,
First you need to optimize fluorescent dye concentration label your sample with a titration of the fluorescent dye you are using after labeling your sample with the fluorescent dye or stain. Wash (in 5 ml tubes containing 3 ml of 1 × PBS) on an orbital shaker (300 rpm, 15 min, 4°C or you can pass at RT also). Washed roots can be transferred to 15 ml tubes (five roots per treatment) containing 2 ml 0.1-0.2%% PBS-S and subjected to sonication using a sonication probe is the right thing to do. The sample can be washed 2–3 times for 10-15 minutes with a buffered saline solution such as PBS is advisable. You can increase your wash time for 20 minutes each with a buffered same solution but 10-15 minute is quite feasible to pass on the results. Incubation with primary Abs at + 4°for 2-3 days then 0.01 M PBS washes twice- thrice each 15 min. These hydrophobic dye molecules will generally bind nonspecifically to tissue sections. To fix the tissue, 4% PFA +0.01% picric acid should be used during perfusion can be organized. In case of reduction of autofluorescence,, ammonium acetate buffer (pH 5) or 1% Sudan BlackB (SB) in 70% ethanol reduced can be useful for auto fluorescence in sections of monkey, human, or rat neural tissue. If you want to reduce tissue autofluorescence, treat the tissue with solutions of similar non-fluorescent diazo dyes.
Ashish
Ashish Thakur and J. C. Tarafdar Thank you for the reply. I will try and see if it will work.
Thank you.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence