Re: [Histonet] Dirty H after break
Hi Allan, The flakes are alum precipitates. I would suggest filtering the haematoxylin before use. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Principal Scientist, the Children's Hospital at Westmead Adjunct Fellow, School of Medicine, University of Western Sydney Tel: 612 9845 3306 Fax: 612 9845 3318 Pathology Department the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: Allan Wang via Histonet [mailto:histonet@lists.utsouthwestern.edu] Sent: Wednesday, 4 January 2017 4:45 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Dirty H after break Hello all, Does someone know what would cause dirty flakes of light purple that showed across the whole slide? http://i.imgur.com/1nIXwAy.jpg Reagents are relatively fresh but the H stainer hadn't been used for a week over the holiday break. Is it surface film on the hematoxylin? I didn't notice anything in the container. We are using Mayer's, followed by differentiation with HCl acid alcohol, with 2-3 minute long wash steps in between. Thanks, Allan ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This message is intended for the addressee named and may contain confidential information. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message are those of the individual sender, and are not necessarily the views of NSW Health or any of its entities. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Dirty H after break
Hello all, Does someone know what would cause dirty flakes of light purple that showed across the whole slide? http://i.imgur.com/1nIXwAy.jpg Reagents are relatively fresh but the H stainer hadn't been used for a week over the holiday break. Is it surface film on the hematoxylin? I didn't notice anything in the container. We are using Mayer's, followed by differentiation with HCl acid alcohol, with 2-3 minute long wash steps in between. Thanks, Allan ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] grad student problem
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
Re: [Histonet] grad student problem
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] grad student problem
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
[Histonet] Dispensing formalin
For those of you working in smaller hospital Histology labs (not staffed 24/7) - How does your OR/Lab handle getting specimens into fixative? We currently use small/med prefilled containers for smaller specimens. Does the OR dispense formalin into larger containers? Do you refrigerate unfixed specimens? How do you handle specimens collected after pathology hours or on weekends/holidays if they are not fixed? I'm just looking for alternative processes in trying to improve ours. Thanks! Terri Terri L. Braud, HT(ASCP) Anatomic Pathology Supervisor Laboratory Holy Redeemer Hospital 1648 Huntingdon Pike Meadowbrook, PA 19046 ph: 215-938-3689 fax: 215-938-3874 Care, Comfort, and Heal ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
