Pine needle-safranin,fast green staining.

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Seb28
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Pine needle-safranin,fast green staining.

#1 Post by Seb28 » Sat Apr 04, 2020 9:34 pm

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

MichaelG.
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Re: Pine needle-safranin,fast green staining.

#2 Post by MichaelG. » Sat Apr 04, 2020 9:43 pm

Looking good, Seb
... What a fascinating structure.

MichaelG.
Too many 'projects'

MicroBob
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Re: Pine needle-safranin,fast green staining.

#3 Post by MicroBob » Sun Apr 05, 2020 1:36 am

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

tgss
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Re: Pine needle-safranin,fast green staining.

#4 Post by tgss » Sun Apr 05, 2020 11:38 am

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.

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Seb28
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Re: Pine needle-safranin,fast green staining.

#5 Post by Seb28 » Sun Apr 05, 2020 3:51 pm

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.
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-Reichert Polyvar
-Olympus IX70
-Zeiss Photomicroscope
-Canon 600D

MicroBob
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Re: Pine needle-safranin,fast green staining.

#6 Post by MicroBob » Sun Apr 05, 2020 4:08 pm

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

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Seb28
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Re: Pine needle-safranin,fast green staining.

#7 Post by Seb28 » Sun Apr 05, 2020 6:02 pm

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
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-Zeiss Photomicroscope
-Canon 600D

tgss
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Re: Pine needle-safranin,fast green staining.

#8 Post by tgss » Sun Apr 05, 2020 7:12 pm

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?)
Last edited by tgss on Sun Apr 05, 2020 8:23 pm, edited 1 time in total.

MicroBob
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Re: Pine needle-safranin,fast green staining.

#9 Post by MicroBob » Sun Apr 05, 2020 7:43 pm

BTW: The documentary images of your sectioning process are exceptional, Seb!

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Re: Pine needle-safranin,fast green staining.

#10 Post by Seb28 » Sun Apr 05, 2020 8:29 pm

Hi Seb
Can you tell us what PEG molecular weight you are using for the hand sectioned samples?
I'm using PEG1500.
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tgss
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Re: Pine needle-safranin,fast green staining.

#11 Post by tgss » Sun Apr 05, 2020 11:10 pm

Seb28 wrote:
Sun Apr 05, 2020 8:29 pm
Hi Seb
Can you tell us what PEG molecular weight you are using for the hand sectioned samples?
I'm using PEG1500.
Thanks Seb. Sorry for wasting your time! I already edited my post to tell myself it was 1500, as stated in your original post. Duh!!
Tom W.

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