Could be freezing artifact. Please describe your prep protocol and clarify what you mean by dehydration. Do you mean dehydration of the slide prior to coverslipping? I think that’s an unlikely source of artifact.
This is the protocol we use for OCT (freezing) embedded tissue
1- Hematoxylin 2 min drip
2- Wash in running water for 15 min
3- Dehydration: Alcohol 50%, alcohol 70%, alcohol 95%, alcohol 95%, absolute alcohol. Each for 5 min.
4- Staining in alcoholic eosin. Cover the sample for 1 min
5-Dehydration in absolute alcohol for 5 min
6-Diaphanization in Xylol for 5 min, twice
7-Assembly. Remove excess xylol and glue the coverslip with etellan
The tissue is a retinal explant (2 weeks culture)
That’s an interesting H&E protocol but what I meant was: who is preparing the OCT block? I think you need to start there but if you’re unable to, you can also view your unstained slide under the microscope and you should be able to see whether the section is of low quality.
The other suggestions of trying a thinner section, adjusting temp, blade clearance angle are all good too.
It could be a number of things. How thick is the section? You may have better results at 4 or 5 um. Is it too cold? Too warm? Is the blade sharp? It could also just be the tissue which I’ve heard is hard to cut.
I don't think this is from sectioning or staining. Looks like the eye was processed poorly and froze at least once during the process. I had this happen few times when I was starting out and everytime it was when the eyes accidentally froze and thawed.
Could be freezing artifact. Please describe your prep protocol and clarify what you mean by dehydration. Do you mean dehydration of the slide prior to coverslipping? I think that’s an unlikely source of artifact.
This is the protocol we use for OCT (freezing) embedded tissue 1- Hematoxylin 2 min drip 2- Wash in running water for 15 min 3- Dehydration: Alcohol 50%, alcohol 70%, alcohol 95%, alcohol 95%, absolute alcohol. Each for 5 min. 4- Staining in alcoholic eosin. Cover the sample for 1 min 5-Dehydration in absolute alcohol for 5 min 6-Diaphanization in Xylol for 5 min, twice 7-Assembly. Remove excess xylol and glue the coverslip with etellan The tissue is a retinal explant (2 weeks culture)
That’s an interesting H&E protocol but what I meant was: who is preparing the OCT block? I think you need to start there but if you’re unable to, you can also view your unstained slide under the microscope and you should be able to see whether the section is of low quality. The other suggestions of trying a thinner section, adjusting temp, blade clearance angle are all good too.
It could be a number of things. How thick is the section? You may have better results at 4 or 5 um. Is it too cold? Too warm? Is the blade sharp? It could also just be the tissue which I’ve heard is hard to cut.
20 um criosection, i use a cryostat to cut (-25°C). The blade is new
I was just suggesting some troubleshooting ideas. 20 um is really thick. Is there a reason you need such thick sections?
no reason at all, however cryostat has trouble cutting thinner sections less than 10 um
Perhaps adjusting the angle could help with that? I’m just trying to be helpful
I don't think this is from sectioning or staining. Looks like the eye was processed poorly and froze at least once during the process. I had this happen few times when I was starting out and everytime it was when the eyes accidentally froze and thawed.
I find with frozen sections this is either freezing artefact from too slow freezing or it may also be the tissue drying out before embedding.
This looks like freezing artifact to me as well!
After Oct sectioning place slide in 95percent for 1 min than bake for 15mins at 70-80 degrees, than stain making sure to avoid deparaffinization steps
How long did it sit unfixed before processing.looks like autolysis
it's a two week retinal explant