I am doing Aniline blue staining in rice anthers to detect callose signaling. When anthers are squeezed and stained, I could detect the signaling. However, when I do plastic embedding with Technovit7100 to get fine sections and do staining I couldn't detect the signaling in any of the different anther development stage. Not sure what went wrong. I fixed sample in PFA/PMEG buffer. Any suggestions to improve my method are highly appreciated.
Thanks in advance!
Hello,
If in understand, you do staining after plastic embedding. Maybe the target is no more accessible to the stain.
Did you try to stain before plastic embedding? This stain may survive to embedding protocol.
Hi Nicolas,
Usually Aniline blue staining is recommended post-embedding, though I didn't give it a try before embedding step. I will try it and let you know if it works.
Thanks
I've plastic embedded plant material into a number of different plastics at different times and stained for callose with aniline blue. Callose is completely accessible to the stain in sections. You just need to adjust your staining time and/or aniline concentration from what you do with the squeezed material. You mentioned neither in your protocol.
Hey Brent!
I tried with both 0.01 % and 0.1% Aniline blue in sections as well as squeezed samples and then immediately observed under microscope . In squeezed samples, both concentration gave signals; however, sections doesn't make any difference in either of the concentrations!
Should I play with time?... How about your protocol?
For this stuff, I got the best results using plant material embedded in MBM (methyl butyl methacrylate) after formaldehyde fixation.
From memory (which isn't what it used to be); 2 to 4 um sections were cut with glass knives and the MBM removed by placing the slides in acetone for 20 minutes. 0.1 % aniline blue for 5-10 minutes, washed briefly, and then sections coversliped using a water based medium used for fluorescence; in my case Fluoromount-G.
I'll try to get some images and the exact protocol when the researcher returns to the dept; he's not in today and he has all the exact details and pictures.
Howdi;
More details as I found the student; 0.1% anailine blue in 0.1M K3PO4 pH 12.
All sections were stained for 30 minutes at room temperature.
4 um Epon sections = no staining.
4 um JB4 sections = good callose signal with some background noise.
3 um Technovit = some signal (not as good as JB4).
3 um MBM = good callose signal with some background noise.
3 ums MBM after MBM removed in acetone = very good callose signal.
From your experience, it can be understand that signal strength varies with different plastic material. So far I used only technovit and I couldn't detect any. Will try other plastics (Preferably MBM) and will get back to you.
Meantime, I am curious to have a look at your pictures of callose embedded with different plastics. BTW, what tissue you've had analyzed for callose ?
The project was to gear up for Yew and Douglas Fir ovule to megagametophyte stages. The testing was done on various plant material that I'd embedded in various plastics over the years. Once we found what worked best we did one run in the plants of interest in MBM. At that point the student took over, did the staining once, and then decided it wasn't of much use to her project and then dropped it, this was 7 years ago. We're having problems finding the images. I'll keep digging through all my back-ups and hopefully find them soon.
B
I am looking forward to seeing the pictures. Catch you soon again
Thanks Brent, that's informative!
Howdi;
Attached is a plate of poplar stem fixed in formaldehyde, embedded into MBM, sectioned at 5 um, sections with both MBM still present or removed, and stained in .1% aniline blue for 30 minutes. (all material s and procedures from Thaumatin-like proteins are differentially expressed and localized in phloem tissues of hybrid poplar. ArticleFull-text available from my site.
The top plart of the plate is with the plastic present and the bottom is another section where the MBM had been removed before staining.
The plate shows sieve plates well stained for callose.
Oh! that callose looks great with MBM plastic after removal. I recently switched the buffer from 4%PFA/PMEG (Earlier) to 4%PFA/Glutaraldehyde (Now) and detected the callose signal (Seems like the buffer was the main culprit ), however, it's similar to the top one in your picture.
Next, I would like to try with MBM and see how good the signal improves.
Thanks and Regards!
Great, post a pic when you have more results.
I've not heard of PMEG buffer before. What is this ?
Cheers
B
Hello there, Sure I will.
PMEG is a buffer which is believed to stabilize the sample well for Immuno-staining particularly. The sample was fixed in 4%PFA/PMEG by one of the post-doc in our lab and I used the same sample for aniline blue staining and it doesn't work. Later I changed buffer to glutaraldehyde and it worked (Yet some background noise).
Cheers!
The attached file is Anther. Sample fixed in 4% PFA/Glutaraldehyde. 3um sections Technovit stained with AB for 30 min. Yet, you can see some background noise.
Excellent !
A bit of background noise is somewhat normal/nothing to be worried about at the level within this picture. A 2 um section may remove the background completely while still giving you enough signal from the anther.
Cheers
B
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology
- Sectioning