Hello fellow researchers,
I am having trouble with H&E staining. My slides end up blurry and unable to focus. I am following what I was taught, samples fixed in 4% PFA overnight, sucrose gradient and frozen in OCT. Slides sectioned at 20, 10, 5, 3um.
For H&E I have tried 10, 5 and latest is 3um (attached problem).
PBS wash, H 1 minute, dip in tap water then immerse in fresh tap water 3-5 minutes, 100% alcohol 30 seconds, E 1 minute, dip 10x in tap water, 100% alcohol 30 seconds, mount with cytoseal 60.
Initially thought sections were too thick so kept scaling down until 3um. Still having the same issue. I have done dehydration with 70, 90, 100% and saw no difference. Suspected residual liquid so I left slides to air dry for 10 minutes before mounting. Did not work either. Tried with one slide to air dry 3 hours, same issue. Cytoseal has toluene.
Anyone knows what I might be doing wrong?
Wash well in PBS before starting to remove the OCT. It looks that maybe they are not properly dehydrated so try 70-90-100-100% ethanol for 1 min each and then xylene at least for 5´.
I hope it helps
I would recommend to omitt the sucrose gradient (which in my experience does not help very much), shock-freeze the tissue in N2 cooled isopentane and cut 5 micrometer sections, which you should collect on silane coated slides.
Best regards, Fred
Mohamed Mahdy
Fred Sinowatz
Our lab has established protocols and I am new so I have been following them.
Samples are placed into OCT then flash frozen in a styrofoam box filled with dry ice and 100% ethanol. It takes anywhere from
I feel the blurriness may be related to retention of moisture than the thickness of the section. While dehydration include two or three additional jars of 100 % ethanol and dip the slides until they do not turn white and foggy when you place it in xylene. I use to experience the same with my FFPE sections in wet season when atmospheric moisture content use to be high. I use to place the xylene jar along with slides at 60 degree for short time to get rid if moisture and mount them after getting cleared.
Pancharatna Katti Hi and thanks. The logic for thickness was, the thinner the sections, the quicker they dry. 10-20um is common for frozen sections but not paraffin, which was why I scaled down to 3-5um. Did not see a difference and I still struggle with the problem, I have done some troubleshooting by increasing length of times for ethanol dehydration (1 minute each, 70%, 90%, 100%, 100%) and clearing with a xylene substitute, even though the mounting media contains toluene, results are the same. I will be storing some samples for paraffin until I eventually (if ever) get the frozen sections to work.
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