I don't know why my paraffin section of fat is like this (as shown in the picture). We can see the cell membrane is not good, the cell is not well-stacked. The adipocyte tissue is from C57BL6/J mice (6 month old).
With standard methods of fixation lipids are largely lost from tissues during processing. If you want to fix lipids you have to render them insoluble with osmium
tetroxide and chromic acid... but I have never done that myself; I'm sure you can google a protocol
With standard methods of fixation lipids are largely lost from tissues during processing. If you want to fix lipids you have to render them insoluble with osmium
tetroxide and chromic acid... but I have never done that myself; I'm sure you can google a protocol
Well, I guess this is a problem with the tissue section, not the processing of the tissue itself. Be careful not to heat to much your sections when cutting them and mounting on slides, or when you heat them to extract the excess paraffin. You could also cut thicker sections, 5 micrometers or larger, to guarantee it will remain integrate.
Good luck!
Is it H&E stain? Fat can easily dissolve during dehydration-rehydration process using ethanol.
We do paraffin-embedding of mammary glands from Bl/6 mice routinely (O/N fix with 4% PFA at RT), and the adipose cells usually have a nice morphology. How do you embed the tissue?
hello, everyone, I am so glad to receive so many good answers. Thanks a lot for all the answers.
Elvira, I want tell you that I fix the WAT with 4% PFA at 4 degree refrigerator for about 2 days. Do your think the time is too long for the fixation?
Their cellular contents are miscible in xylene (aswell as ethanol) and by the time you've embedded, dewaxed and then cleared before mounting cover slips majority of adipocytes become devoid of cellular contents.
I think this is a normal thing happened with fatty tissues when embed it, most of the lipid lost because it is a harsh method using Xylene and ethanol so when fatty tissue within the muscle you can keep the structure but when there is a large amount of fat you will face this thing, I worked with breast cancer also contains fatty tissue but the area with fatty tissue mostly lost during processing, may openion is the fatty tissue and you have to do it many times then you can manage which is the better method
Good Luck
Cryostat has its own problems, you're quite likely to crack the tissue due to the lipids rigidity when frozen. If you wish to analyse lipid composition histological imaging is not the best way forward, try cell lysis and reverse phase chromotography.
Actually I find it's not so bad your sections! you migt get improvement of tissue architecture if you use perfusion fixation of the mice before sacrifice, and 2 days in 4% PFA certainly seems reasonable, but you should also consider the SIZE of the tissue pieces, where smaller is always better for penetration. During embedding I increased bath time (as compared to traditional parafin embedding) in the various solvents and paraffin steps to get complete penetration, but perhaps you already do that, because I think your adipocytes looks quite fine. Once your sections are cut, do you just hydrate them and then stain H&E? Perhaps some additional fix before this step could be considered to hold 'flimsy' cell membranes in place. test with 10 min 4% PFA or 20 min 100% acetone, which will remove all remaining lipid drops, then follow with rehydration and stains. Could you please upload photos any of the suggestions you have gotten leads to an improvement? would be nice to see!
I think you can try fixing the fresh tissues in 10 buffered neutral formalin for 72 hrs at 37oC and then go for routine paraffin processing technique for H& E staining of fat cells. this will give you the normal morphology of the adipocytes bu with an empty cytoplasm, if you want to preserve the cytoplasm you can opt the standard text protocol (Animal tissue Technique- by G.L. HUMASION).
This is the normal appearance of fat cell when fixed in neutral buffered formalin.
If u want to see fat droplets, fix in bouin's fluid, and stain with sudan III stain, or use crystate to make frozen sections.
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