Dear all,
We are currently attempting to capture images of cells on coverslip cross-sections in the lab. Here is the protocol used for processing the samples:
1% OsO4 - 2% ferrocyanide (in 0.1M cacodylate) staining for 30 min at RT
1% OsO4 (in 0.1M cacodylate) staining for 30 min at RT
1% uranyl acetate (in ultrapure water) staining for 30 min at RT
successive ethanol dehydration (25% ethanol for 10 min - 50% ethanol for 10 min - 70% ethanol for 10 min - 95% ethanol for 10 min - 100% ethanol for 3x10 min)
propylene oxide dehydration for 2x15 min
incubation in 50/50 propylene oxide/AGAR100 for 1h30
incubation in AGAR100 for 1h30
Then we change the AGAR100 and mount the coverslip on a beem. The resin is left for 2 days at 60°C.
Our problem is that we have observed a lumpy texture during the observation session - see image 1. Does someone know where it can come from?
(We suspect a problem during the dehydration of the sample, but this protocol has been used several times in the lab.)
Also, during the sectioning at the ultramicrotome, we observed that the cells were textured (not smooth in appearance) and sometimes thicker than the resin itself (the cells were blue, and the resin gray) - see image 2. Once more, does someone know where it can come from?
Thank you in advance,
Best regards,
Erika
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The lumpy texture in your TEM samples could be due to incomplete dehydration or resin infiltration. Rapid ethanol transitions may cause osmotic stress, leading to uneven embedding, while improper polymerization can result in textural inconsistencies. You might consider longer AGAR100 infiltration or vacuum-assisted embedding to improve resin penetration. Also, switching to acetone for dehydration could enhance results. For those interested in material durability, shingles commercial 2025 might be a useful reference.
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