I am trying to visualize alpha and dense granules in platelets. However, it seems like the granules were not stained well. Is there any way I can improve my staining technique to obtain better contrast for better looking images? Below is the staining technique I have used:
1. Uranyl acetate (15 min)
2. Ultra Pure water (3 min)
3. Ultra Pure water (1 min)
4. Lead citrate (7 min)
5. Ultra Pure water (2 min)
6. Ultra Pure water (2 min)
7. Let dry for TEM
Uranyl acetate and Lead Citrate were filtered with 0.22 micron syringe filter prior to use.
Lead citrate was prepared by dissolving 5mg of lead citrate in 1mL of 4.5mM NaOH.
Is it better that I prepare lead citrate using sodium citrate and lead nitrate?
Attached are some of my TEM images from resting platelets. Thank you in advance!
Hello,
It is the protocol for contrasting the sections.
Did you contrast your cells after fixation, with osmium tetraoxid (followed by optional uranyl acetate) before dehydration and resin embedding?
After fixation, I would recommend staining your cells for 1hr at 4C with 1% osmium tetroxide in 0.1M Cacodylate buffer (pH=7.4). Also, to improve the contrast and ultrastructural visualization, overnight night en-block staining with Uranyl Acetate (1-2%: W/V; dissolved in double distilled water) can be very helpful. In this way, most likely, you wouldn't even need to post-stain your cells with Lead citrate.
Good luck,
Reza
Thank you for your reply Nicolas Barois , yes, platelets were fixed in 2% glutaraldehyde and 2% formaldehyde in 0.1M sodium cacodylate buffer, followed secondary fixation in 1% OsO4 in 0.1M sodium cacodylate buffer in room temperature for roughly 2 hours. I will visually check for the even darkening of the platelets. Then I will wash away OsO4 with ultra pure water, followed by 1% uranyl acetate for 1 hour. After that I will then proceed with dehydration and resin embedding.
After I obtain the grids I also conducted the staining steps as mentioned above, again with uranyl acetate as I am still trying out. Please suggest me if any step should be added or omitted.
Thank you Reza Khazaee may I know does en-block staining mean that after embedding my sample in resin, I should immerse the whole resin in 1% uranyl acetate overnight before microtome sectioning? Thank you in advance!
en-block staining: after washing off the Osmium, and before dehydration, immerse the samples in freshly made filtered 1-2% UA for overnight in the fridge. How long you spend for each dehydration step? Based on my own experience, each dehydration step should take between 10-20 min.
Also, you can get cleaner results by doing Osmium staining at 4C, Instead of room temperature.
By the way, do you use freshly-made fixative? Old fixative (more than 1-week old) can affect the results.
Reza Khazaee thank you for your suggestion! I will try to do en-block staining in UA overnight at 4C. My dehydrations steps were also 10 mins each.
The fixative I used is slightly more than a week old. I will also try with freshly made fixative. Thank you so much again for your useful suggestions!
Yuan Yee Lee your protocol looks fine for me. I can guess structures inside cell but everything is too dark, not enough contrast. Membranes can't be seen. Too much staining?
As you have osmium and uranyl in block, you can look at your sections without staining (no uranyl and lead staining on sections). The camera is much more sensitive than eyes and you will see your sample (personally I am not friend with the lead citrate, I never contrast my sections).
You’re welcome Yuan! I agree with Nicolas; with en-bloc staining usually there is no need for post-staining on sections
Reza Khazaee Nicolas Barois got it! I will try without post staining on sections. Thank you for your suggestions, I appreciate it!
Hi Yuan, I think the problem is with fixation. Please watch out for fixatives and respective procedures as discussed above.
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