could you please help with a protocol?
should i do it directly on the aluminium stub after dehydration with 100% alcohol or not?
should i release the pollen or whole anther is better and dehisce it later?
after drying with HMDS how can i keep the specimens on aluminium stub before coating for about 24h?
Drying Samples for SEM With Hexamethyldisilazane
This method was designed to dry previously fixed and dehydrated SEM samples without using a critical point drying device before sputter coating and viewing with an SEM.
Procedure:
1) fix tissue by routine methods
2) Dehydrate tissue to 100% ethanol using a graded ethanol series
a) Buffer wash, twice, 5 minutes each
b) 50% ethanol, 10 minutes
c) 70% ethanol, 10 minutes
d) 80 % ethanol, 10 minutes
e) 90 % ethanol, 10 minutes
f) 95 % ethanol, 10 minutes
g) 100% ethanol, 10 minutes twice
3) Remove 100% ethanol and replace with hexamethyldisilzane. Do not let the hexamethyldisilzane come into contact with skin; it is TOXIC. After 5 minutes, replace with fresh hexamethyldisilzane.
4) Place specimens in hexamethyldisilzane in a glass Petri dish at room temperature in a fume hood. Partially cover the dish with its lid.
5) After the samples are completely dry (usually overnight), mount them on SEM stubs, sputter coat them, and examine them with the SEM.
dear Brent E Gowen thanks for your answer.
-Can i keep the samples in 2.5% Glutaraldehyde in PBS Solution for 30 days before dehydration?
-For buffer wash before using graded ethanol, you mean PBS buffer?
-I use 2X2cm aluminum sheet pieces as stub, at final step can i put the anthers on aluminum then in the glass petri and HMDS? just for better stick on aluminum stubs.
it is not ideal, but you can keep your samples in a 2.5% glut for 30 days before dehydration. Just make sure the samples are in a fridge
yup, buffer wash = whatever buffer your fixative used was in.
Dry your sample, then adhere it to your stubs with double-sided tape. I don't think your samples will stick to the stub through the process. I've used poly-l-lysine to get samples to stick to a glass coverslip. On such a coverslip, the samples will remain adherent throughout the processing. You should look into that.
Dear Brent E Gowen ,
As you mentioned that 2.5% glut in PBS is to ideal to keep samples for longer time, to prevent damage and to get best result is it better to continue the process and doing dehydration with ethanol but stop at 70% grade(samples in vial includes 70% ethanol) and continue it after 30 days?
- When i place the specimens at glass petri should i add hexamethyldisilazane for third time? if yes, how much?
-At the final step when the specimens rest in the hood overnight is it necessary to put silica gel at the hood for humid absorption?
I have done both for similar samples (plant material); left in glut for over a month or dehydrated to 70% ethanol and then left for over a month. Ultrastructurally by TEM, I found the material to be better left in the glut. There were some extraction artifacts from the material stored in 70% ethanol that I did not see from the material stored in glut.
I've stored fixed material, after dehydration to 70%, for a few days without extraction artifacts. You could try both methods for your sample. Maybe by SEM you won't see a difference and then you'll know what to do in the future.
Yup, hexamthyl. for a 3rd time. I have small glass petri plates (you must use glass for this...not plastic). I just over the samples with hexamthyl and have the dish cover over the bottom at an angle so that the hexamethyl can slowly evaporate. I've not put silica gel in the hood for humid absorption.
B
I've used both double-sided Carbon tabs and tapes. If you have double-sided aluminum adhesive, I'd imagine that might be better as it is probably more conductive within the SEM than the tapes and tabs and so a lighter/thinner sputter-coating can be applied to the sample.
B
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