Completely agree...it sounds like the tissue got freeze dried and I doubt if any staining you might get after lots of work will be completely reliable.
Loralei On Jan 3, 2017 11:02 AM, "Caroline Miller via Histonet" < histonet@lists.utsouthwestern.edu> wrote: > I hate to say it but I think these tissues are toast. It seems they had bad > fixation or handling to start with, and I would question whether the > tissues were left out to dry before fixation or freezing. Or whether they > got fixed and then sucrose infiltrated (which is my method of choice if the > antibody allows) > > Also - what type of tissues? If it is lymph nodes then maybe there is too > much fatty tissue and that is why it is not sectioning. > > I have had a hard time with T-cell markers in paraffin so I do see the > hesitation in paraffin embedding. If you do go that route then certainly > thaw in formalin, not straight water. > > Not much help, sorry! But sometimes it is just better to go back and plan > the experiment properly in the first place than trying to mess around with > old bits of tissue - because even if you could get it on the slide, would > you really trust the results of the immunostaining??? > > Happy New Year All! > > yours, > mills > > On Tue, Jan 3, 2017 at 10:05 AM, Roberta Horner via Histonet < > histonet@lists.utsouthwestern.edu> wrote: > > > I got the following from a grad student here at Penn State. I am not sure > > how to solve his problem if possible. Does anyone have any suggestions I > > can forward? > > Roberta Horner > > Animal Diagnostic Lab > > Penn State University > > > > "I am having some difficulties sectioning mouse tumor samples for > > immunofluorescent analysis. We originally went the OCT route because we > > are staining for T cell markers and were worried that the heating that > > occurs during paraffin embedding would compromise the T cell receptor. > The > > samples are a little old, but we are hoping to section and stain for > immune > > cell infiltrates. When sectioning with the cryostat, the tissue and OCT > is > > quite brittle and the sample is not intact enough to transfer to a slide. > > Two colleagues have given the following suggestions: > > 1. Thaw the OCT blocks, remove the tissue, and place in a new block with > > fresh OCT media. I've tried this technique on a few practice samples and > > the new OCT media seems to be less brittle and I'm able to get tissue on > a > > slide, however the tissue itself still seems to be poor with either > freeze > > or sectioning artifacts. > > 2. Thaw the OCT blocks in water, remove the tissue and place in formalin > > overnight, place in 70% EtOH, then paraffin embed. Section on a > > microtome. Check the fluorescently labelled antibody data sheet to see > if > > paraffin embedding interferes with binding. Try to stain and see what > > happens. > > > > I was hesitant to try the second suggestion because I have found no > > protocol that takes tissue originally stored in OCT blocks and > subsequently > > redirects them to formalin and paraffin for microtome sectioning. If you > > have any recommendations on how to move forward and section these > difficult > > samples, or know anyone at the diagnostics lab or at Penn State that > could > > help, that would be much appreciated!" > > > > _______________________________________________ > > Histonet mailing list > > Histonet@lists.utsouthwestern.edu > > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > > > > > -- > Caroline Miller (mills) > Director of Histology > 3Scan.com > 415 2187297 > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet