Is fat cell looked white after HE staining? - (Oct/05/2008 )
Hi, all,
I am doing Hemotoxylin&Eosin staining of mice muscle now. My samples were cryofixed in OCT and stored in -80C. First I cut 20 microns sections, then do HE staining. But I am confused how to distinct fat cells with holes in the section. Is fat cell white after HE staining or does it have pinkish cytoplasm visible? It seems to me holes within muscle section are white under microscope. Please help me out, thank you in advance!!
Yes, fat cells do look like holes.
Would you post the picture of the tissue section, please?
Would you post the picture of the tissue section, please?
this is picture by my staining. I am not sure if it's uploaded.
I have put 3 pic on a PPT file, Could somebody have a look and give me a clue? Thanks
Pic B, C were stained by our lab tech, but she is gone to another town. Pic A is done by me. I used acidic alcohol and Scott's tap water, but she didn't.
Do the tissue sample slides come from the same tissue block?
These don't look like fat cells to me, they look more like tears (breaks) in the section. Fat cells look like cells with round edges. google images "Adipose tissue section" should give you some idea of the sort of thing you would expect to see.
I would also recommend lowering the eosin staining time a bit, your sections are looking pretty pink.
No, they are from different mice back muscle(I cut a spinal cross section).
My purpose is to see if the intramuscular fat is different between treatments. Don't know which staining is more appropriate, Oil-red O, HE or some others?
I would also recommend lowering the eosin staining time a bit, your sections are looking pretty pink.
Thank you for your advice! I also doubted if those white shapes are fat cells, but my boss(obviously not a histology pro) said since there are blue dots at the edge of white shapes(nuclei), they are fat cells(See pic
.I would do more comparison to convince him they are not.
Could you please tell me a bit more how to avoid tears during cutting? I did section on a pretty old mirotome,
-20C, and 20 um thickness.
Would 4% Paraformaldehyde fixation, parafin embedding section be better than cryosection in terms of muscle cell and fat cell staining?
Any input will be greatly appreciated.
-20C, and 20 um thickness.
Please see this video on frozen sectioning on microtome.
http://www.bio-alive.com/community/play.php?vid=112
Paraffin sections would definitely be better and easier, in my opinion, but I am not a histologist.
I have attached a picture of a fat pad section from a mouse:
[attachment=5400:FAT.png]
I have labelled the different types of tissue you can see in the section. The white fat (which is what you will see in the muscle) is in the bottom left of the picture. Stroma is just underlying connective tissue. The eosin is a bit faded as I just use these slides for setting up microscopes, so they sit on my desk in the light. Note that the fat appears as an empty white space because the use of solvents such as xylene has removed all traces of the fat. I am not sure how cryosections are processed, but if you use xylene or other non-polar organic solvent then there won't be any fat left to stain.
I would really recommend getting a good histology/immunohistochemistry textbook from the library.