Hello scientific community on Research Gate,
It has been a rough week as I have been troubleshooting to resolve mediocre paraffin sections.
Initially, I thought it was due to improper fixation (inadequate 10% NBF recipe) of my specimen. Then, I was made aware that absolute EtOH is not available in our lab and we were using 96.6% EtOH as a substitute. Therefore, it was sensible to assume that incomplete tissue dehydration could account for the observed artifacts on the paraffin ribbon. I managed to find some 99.9% EtOH and repeated the procedure, but to no avail.
It seems that the microtome blade, that I replaced, is cutting rather around (and not through) the tissue which leaves a perforation on the ribbon corresponding to my embedded specimen. On better instances, I would manage to obtain a (semblance of) section of my specimen with noticeable lacerations and tears.
My hunch is that the paraffin has deteriorated (expired since 2008) and is poorly impregnating the specimen.
As per your anecdata, what is causing this horrendous (at best) paraffin section?
Is this artifact most likely attributed to processing (i.e., fixation, dehydration, clearing, infiltration) or defective material?
To guide your diagnostic input, here's an approximate description of the protocol:
Fixation
10% NBF - 48h
Dehydration
60% EtOH - 1hr
70% EtOH - 1hr
80% EtOH - 1hr
90% EtOH - 1hr
100% EtOH - 1hr
(All of which are calculated/prepared from a 96.6% EtOH bottle deemed as 100%)
Do you think I am being overzealous to procure 100% ethanol or the available 96.6%EtOH would suffice for dehydration?
Clearing
Xylene - 1hr
Wax infiltration
In paraffin (Overnight, oven @70°C).
P.S. I have included snapshots (1, 2, 3 — from bad to worst) of the modest chef-d'oeuvres that I've produced for the past 2 weeks and it's borderline frustrating.
Your compassion and expertise are greatly appreciated.
Thank you!
Hello Sami,
If you look at the results of a broken or perforated cut in the organ, I assume several causes including:
1. Organs are too hard or too soft, this is related to the process of dehydration and the process of clearing. For the dehydration process I suggest the following protocol
70% alcohol @ 30 minutes x 4 times
Alcohol 80% @ 30 minutes x 2 times
Alcohol 90% @ 30 minutes x 2 times
Alcohol 96% 30 minutes
For the clearing process, you can try using a toluol solution for 2-5 hours. This depends on the type and size of the organ.
2. Paraffin does not enter the organs properly, this is caused by the infiltration process that is not optimal. For the infiltration process I suggest the following protocol.
Paraffin : toluol (1 : 1 ratio) for 30 minutes
Paraffin for 60 minutes
Paraffin for 60 minutes
Paraffin for 60 minutes
3. Cutting technique, in this case you only need to practice frequently and routinely check the condition of the knife using a microscope.
Hope this helps you,
Regards,
Sandi Fransisco Pratama
Hello Sandi Fransisco Pratama,
I sincerely appreciate your thorough feedback and insightful suggestions.
I will apply the modifications you proposed on my next trial and will certainly keep you informed of the progress and outcomes.
Thank you for your valuable assistance.
Best regards,
Sami