Pine needle-safranin,fast green staining.
Pine needle-safranin,fast green staining.
After many attempts I managed to stain the pine needle with safranin and fast green. This is not what I expected but so far this is the best result. I have to keep trying to get better results. The pine needle was fixed in FAA and then embedded in PEG1500 for better cutting.I cut the needle under a stereo microscope.
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-Reichert Polyvar
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
Re: Pine needle-safranin,fast green staining.
Looking good, Seb
... What a fascinating structure.
MichaelG.
... What a fascinating structure.
MichaelG.
Too many 'projects'
Re: Pine needle-safranin,fast green staining.
Hi Seb,
that is a nice looking section! What like especially at pine needles ist that you often have clearly visible stomata in the image. I have no experience with PEG embedding - how do you use this and how are your experiences? Does it section well without crumbling? With what type of microtome did you do the section and what thickness did you cut?
Some ideas for further experimenting:
- Just use a carrot to embed the needle
- Try Etzold FCA or Wacker W3A stains - I think they stain different tissues more differentiated.
- Try a free hand cut for comparison - needles are fairly easy to cut free hand
Bob
that is a nice looking section! What like especially at pine needles ist that you often have clearly visible stomata in the image. I have no experience with PEG embedding - how do you use this and how are your experiences? Does it section well without crumbling? With what type of microtome did you do the section and what thickness did you cut?
Some ideas for further experimenting:
- Just use a carrot to embed the needle
- Try Etzold FCA or Wacker W3A stains - I think they stain different tissues more differentiated.
- Try a free hand cut for comparison - needles are fairly easy to cut free hand
Bob
Re: Pine needle-safranin,fast green staining.
Very nice result indeed Seb. From your description it sounds like you cut the section by hand. If so you did an excellent job.
Tom W.
Tom W.
Re: Pine needle-safranin,fast green staining.
Thank you all for the comments.
The embedding procedure in Peg1500 is as follows:
After fixation in FAA, I transfer the sample to 20% PEG solution for several hours, preferably 24 hours, then transfer it to 90% PEG solution for several hours, and then transfer the sample from 90% PEG solution straight to the slide and allow it to dry for several hours. I cut the PEG embbeded sample under a stero microscope. After cutting, I transfer the slices to water to dissolve the PEG. Then staining.
The embedding procedure in Peg1500 is as follows:
After fixation in FAA, I transfer the sample to 20% PEG solution for several hours, preferably 24 hours, then transfer it to 90% PEG solution for several hours, and then transfer the sample from 90% PEG solution straight to the slide and allow it to dry for several hours. I cut the PEG embbeded sample under a stero microscope. After cutting, I transfer the slices to water to dissolve the PEG. Then staining.
-Reichert Polyvar
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
Re: Pine needle-safranin,fast green staining.
Hi Seb,
that is a very interesting technique - I have never heard of anybody doing this! Typically people who embed into some media do the cutting with a real microtome. People who do freehand sections don't embed. Your method makes hand sectioning usable for several more applications.
I recently started to do do paraffine embedding and sectioning. One aim was to make slides like crosscuts through flower buds. Do the sections in PEG hold together well enough for a flower bud crosscut?
Bob
that is a very interesting technique - I have never heard of anybody doing this! Typically people who embed into some media do the cutting with a real microtome. People who do freehand sections don't embed. Your method makes hand sectioning usable for several more applications.
I recently started to do do paraffine embedding and sectioning. One aim was to make slides like crosscuts through flower buds. Do the sections in PEG hold together well enough for a flower bud crosscut?
Bob
Re: Pine needle-safranin,fast green staining.
Hi Bob
I haven't tried to embbeding the sample into PEG yet and cut on a microtome, I am going to try it soon but I know that this technique is practiced but in harder PEG for example PEG4000.
Read this article.
https://scholarworks.uni.edu/cgi/viewco ... ntext=pias
I haven't tried to embbeding the sample into PEG yet and cut on a microtome, I am going to try it soon but I know that this technique is practiced but in harder PEG for example PEG4000.
Read this article.
https://scholarworks.uni.edu/cgi/viewco ... ntext=pias
-Reichert Polyvar
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
Re: Pine needle-safranin,fast green staining.
Hi Seb
Can you tell us what PEG molecular weight you are using for the hand sectioned samples?
Regards
Tom W.
Edit: OK - brain fade - I just re-read the post - dilute 1500 is the answer! (Or is it 42?)
Can you tell us what PEG molecular weight you are using for the hand sectioned samples?
Regards
Tom W.
Edit: OK - brain fade - I just re-read the post - dilute 1500 is the answer! (Or is it 42?)
Last edited by tgss on Sun Apr 05, 2020 8:23 pm, edited 1 time in total.
Re: Pine needle-safranin,fast green staining.
BTW: The documentary images of your sectioning process are exceptional, Seb!
Re: Pine needle-safranin,fast green staining.
I'm using PEG1500.Hi Seb
Can you tell us what PEG molecular weight you are using for the hand sectioned samples?
-Reichert Polyvar
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D