Fresh leaf, stem, root, flower bud samples for FESEM
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Notes: Pipes buffer works better than NaCaCodylate buffer - - gives less shrinkage and less distortion. Using GTA/PF in Pipes buffer for several days to a week works great. Eliminating the post-fix Osmium works very well to limit shrinkage and collapse of tricomes. Long dehydrations in 100% ethanol (ETOH) is necessary for complete dehydration. Use a minimum of a 20 minute purge during critical point drying (CPD).
Coat: OsmiumPlasmaCoater(OPC) 6nm or Pulse coat with Sputter coater, using AuPd source, for 3 minutes.
1. Cut small samples from whole leaf, stem, root or use whole flower bud and fix: 3% GTA/1%PF in 0.1M PIPES pH 7.0-7.2. (2ml 70%GTA, 3ml 16%PF, 17ml dH20, 23ml 0.2M PIPES). Put under gentle vacuum for 24 hrs, until samples settle. FIX samples for a minimum of 4 days with several fix changes to harden the botanical samples. At this point you can further trim the samples for clean cross sections or smaller sample size.
2. Remove fixative and wash 3x with in 0.1M PIPES with 0.15M Sucrose pH 7.0-7.2 over 2 hours on rotator at room temperature.
3. Add a 2x water rinse and en-bloc stain with UAaq for 30 min at RT if you want to use back-scattered detector to see nucleus of cells.
4. Dehydrate for 30 min in 30, 50, 70% ethanol (EtOH) solutions.
5. Leave in 70% EtOH overnight in fridge.
6. Wash 3x 30min each in 100% EtOH at RT. Then leave overnight at RT rotator to remove all water from samples. You should leave in 100% EtOH for several days, with a number of fluid changes, to be sure of full dehydration.
7. Critical point dry at this step. Use a 20 minute purge to be sure all the ethanol has been replaced by the LCO2. I have not had a good success drying botanical samples with HMDS.
8. Mount samples, using Apiezon wax
9. Sputter coat 1-2 minute with AuPd and then OPC – 6nm; or just OPC – 6nm or just Pulse coat with Sputter coater, using AuPd source, for 3 minutes.