Hi there! I need advice on a protocol for mouse tissue staining. I will dissect mouse livers and fat pads and look for liver steatosis and adipocyte perimeter. I was planning on doing this with H&E staining, is this the best way to see liver steatosis and adipocyte perimeter?
Once I take out the livers and fat pads, should I put them in 4% PFA for 24 hours and then can I transfer them directly to 70% ethanol for storage for 3 weeks (I am leaving school for winter break and doing the tissue extractions before break and doing histology when I return). Thank you so much in advance!
C. Juneja Chloe Henderson
Best variant is to fix tissue in big volume (over 20-25 volumes of tissue) of 4% phosfate buffer formalin (pH over 7.4) for 24-48 hours and then do histological embibition of tissues. Better leave parafin blocks before winter breaks.
Ethanol for fixation is not a good idea especially for a long period. Ethanol dries tissues and H/E stained tissues will be with artifacts.
To my knowledge, tissue fixing for H&E staining dissolve out lipids. Therefore, it would be quite strange if you saw lipids in an H&E stain embedded in a paraffin section.
I would use Oil Red O staining to visualize fat.
Make cryosections at 5-8 um thickness and stain with Oil Red O (These protocols are easily found in web).
Cheers
Susara
Susara Madduma Hewage Thank you so much for the advice! Do you know if this staining has to be done immediately or can I store the liver somehow in a solution (would 70% alcohol work?) and do the staining 3 weeks later? Thank you!
Dmitry Zinovkin Thank you so much for the advice! Would you recommend any other solution that I can store the livers in for a few weeks since I won't have enough time to do the paraffin embedding immediately? Thank you again!
Hi Chloe,
If you do Oil Red O staining, the tissues can be stored at -80C for years.
Whenever you are ready to do the experiment take the tissues out and do the sectioning using a cryotome.
Remember..! this is only for cryo-sectioning.
For paraffin sectioning we usually dip the tissues in 10% formalin buffer ( 0.6g NaH2PO4 + 1.28g Na2HPO4 + 20ml Formalin + 180 mL tap water ) and use to make paraffin blocks within 2-3 weeks (Make sure to dip the entire tissue in formalin). I haven't try keeping tissues in formalin more than 3 weeks.
Good Luck..!