The attached images are ultrathin sections of a cell and whole embryo of a filarial worm stained with uranyless and lead citrate. I have fixed/processed/imaged similar samples dozens of times with the same protocol with good results, however this time around I noticed a very odd appearance of my sections. The areas of the grid covered by my section appear almost porous, while the formvar where no section is present looks normal. All of the samples I processed from this batch look like this.
The only difference in how these samples were handled compared to previous samples is that these samples were transported in 70%EtOH at variable temps (sometimes pushing 90 F) through mass transit for about 2 hours. I have never processed on ice so I also do not really believe that to be the issue.
Could this appearance simply be due to poor resin making/quality? Maybe an issue in the infiltration of the samples? I used Spurr low viscosity embedding resin as I normally do.
Any thoughts are very much appreciated!
Dear Dr. R. Peguero,
it is just a proposal: would you mind to first provide way better images than you attached to your question (which is - clearly - an interesting one...should be glad to receive your protocol for staining grids with Uranyless as well as the Pb-citrate-stain you use. I understand that you stain your grids manually instead of using an automated GRID-STAINER).
The images provided are blurry and of low resolution and cannot aid an exact interpretation by zooming in.
From what you describe I guess that not the '70%EtOH' treatment is the problem.. but we'd need more detailed information - in general - regarding the specimen processing steps you performed.
There is a possibility that the formvar film (BTW, which solvent did you use when fabricating the film =mounting your grids ?) got some altered microareas which are prone to become porous or develop holes under the ultrathin sections during processing the grids when staining them....(e.g. different surface tension forces during staining and/or washing and drying mode), but first of all:
Please provide HR-images of representative areas of 'odd appearances'. Thanking in advance,
Wolfgang H M.
Yes, I agree with Dr. Muss, the quality of the images are not useful. But if these pores in the section are due to infiltration issue:
- Dehydrate your samples sufficiently. Use 50%,60%,70%,80%,90%,100%,100% ethanol/acetone for dehydration.
- Prepare, store and use resin in the moisture free area.
- Before using resin, make sure there should be no bubble or moisture, reheat the resin in oven for 15-20 min till it gets warm.
- You might need to perform 3:1, 1:1,1:3 and 100% resin steps for little longer.
- You can see thick sections under LM to get the overview of quality of sections.
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