Re: [Histonet] cryostat cutting problems
It may help to let your block equilibrate for about 20 minutes in the cryostat. I've found blocks do not section well if you start sectioning right away, especially snap frozen ones. Also, do you embed in OCT/sucrose? A 1:1 OCT: 30% sucrose solution is much softer than just OCT (or whatever embedding medium you are using). What do you mean by the tissue is condensed? It's folding up? I've found sectioning fresh unfixed tissue on a cryostat is impossible (we tried chick embryo trunks, which may be harder to section fresh than other tissue)--it doesn't section but get smushed to a pulp. You need to use a sledge microtome, unfortunately. What thickness are you sectioning at? I've found sectioning above sixty microns doesn't work as well on a cryostat, but again this is on fixed chick embryo tissue. If your cryostat is old, I would suggest you look for a new anti-roll plate made of glass. They work so much better. As long as your cryostat isn't older than about ten years, you should be able to find one that fits your knife holder very easily. That said, I've used a Rei-something cryostat that was 15 years old and it sucked. The anti-roll plate holder was too loose to do anything useful and the adjustment of the blade holder was impossible. Can you use a newer cryostat in your department from another lab? Or just go back to sledge microtome sectioning--if you know how to do it, it's the same principle anyway. Emily -- Outside of a dog, a book is man's best friend. Inside of a dog it's too dark to read. --Groucho Marx On Fri, Aug 13, 2010 at 4:53 PM, Entwistle, Laura lentwis...@ucsd.eduwrote: I am new to using a cryostat and am having some issues with my tissue. 1.Every slice seems to shatter and fragment. When I have used a microtome it was because the tissue was too cold. 2. The tissue itself was flash frozen and so was not fixed and the tissue did not go through the perfusion process. 3. Each slice is condensed and doesn't lay flat. 4. The cryostat is old. Any tips would be very helpful. ___ 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] please enter
Please feel free to ask questions. I always do because I'm not in pathology (which most people here are) but neurobiology. Emily -- Outside of a dog, a book is man's best friend. Inside of a dog it's too dark to read. --Groucho Marx ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: SPAM-LOW: [Histonet] Staining Racks
We managed to get racks from Leica that fit onto a different HE stainer and the Leica coverslipper I would check with your Leica rep about this. Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos Brooks Sent: Friday, August 06, 2010 7:24 PM To: histonet@lists.utsouthwestern.edu Subject: SPAM-LOW: [Histonet] Staining Racks Hi, I recently acquired a used coverslipper (YEAY ME!!!) It is a Leica CV-5000. I know there are a number of big fans of this coverslipper here, but I have a possible problem. I also have an automatic slide stainer that I LOVE! The stainer is a Sakura DRS-2000. It has a slide rack holder that holds 2 Winlab 20 slide racks (the dusty charcoal colored racks). The Leica takes a rack that holds 30 or so slides with a hanger on either side. This of course doesn't fit the stainer or the stainer rack holder. So here's the conondrum, how do I get these instruments to play nicely together? Is there a rack holder that I could get for the Sakura stainer that would hold the Leica racks? Alternatively, is there any way to use the Winlab racks for the Sakura on the Leica coverslipper without having the slides jam? (coverslippers are ALL finicky) Are there any alternative solutions anyone has found? I had considered fabricating a stainer rack holder that would hold the Leica racks with plexiglass perhaps, but if there is a solution that is already being used I'd love to save the experimentation. I certainly won't transfer the slides between racks after staining. I might as well hand coverslip in that case. Thanks in advance for any help! Happy Friday, Amos ___ 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] please enter
C4d Immunoreactivity has been used to assess transplant failure in kidneys and hearts Peritubular capillary deposition of C4d has been shown to be associated with both acute humoral and vascular rejection and increased graft loss (7,8). This was found to be independent of histological rejection type (7). Patients were considered C4d positive if 25% of the peritubular capillaries exhibited circumferential staining (7). Successful staining relies on Microwave antigen retrieval. Peritubular capillary C4d deposition in acute allograft rejection is a predictor of long term graft failure (7) and it has been suggested that these patients would benefit from intensive therapy, potentially preventing the previously reported high graft failure rate (8). 7. Herzenberg, A.M., Gill, J.S., Djurdjev, O., Magil, A.B., (2002) C4d deposition in acute rejection: An Independent Long-term Prognostic factor J Am Soc Nephrol 13(1):234-41. 8. Nickeleit, V., Zeeiler, M., Gudat, F., Theil, G., Mihatsch, M.J., (2002) Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications: J Am Soc Nephrol 13(1):242-51 Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of mohamed abd el razik Sent: Saturday, 14 August 2010 3:46 AM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] please enter Dear histonetters i have a requist for all of you as i'm new in histochemistery and IHC as many other frinds i know in histonet . we found it difficult to know the name and use of many antibodies in many many topics so we can't understand the all masages and replaing on it. and lose the opportunity to learn from it!! and we are ashamed to talk about that with experts like you.but the group lose its functionality in teaching IHC for the new histologiest sector. so i put our proplem on your hands and my hope is to help us to find solution an example of what i'm taking about is what is C4d and for what it is used? thanx in advance to your help and for these amazing group mohamed Faculty of Vet. Med. Cairo Univ. - Egypt We use the same antibody from Cell Marque on the Dako Autostainers with great success. Loralee McMahon, HTL (ASCP) Immunohistochemistry Supervisor Strong Memorial Hospital Department of Surgical Pathology (585) 275-7210 From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Houston, Ronald [ronald.hous...@nationwidechildrens.org] Sent: Friday, August 13, 2010 10:29 AM To: 'Martha Ward'; histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: C4d on paraffin sections We use the C4d from Cell Marque now with great results; it is an IVD. ER1 20 minutes on the BondMax and Bond III Ronnie Houston Anatomic Pathology Manager Nationwide Children's Hospital Columbus OH 43205 (614) 722 5450 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Martha Ward Sent: Friday, August 13, 2010 9:46 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] C4d on paraffin sections I need some advice. We have been trying to do C4d on paraffin sections without much success. Does anyone have a good protocol we could try? We use the C4d from Quidel for our frozen sections and ideally I would like a procedure that we can use with our Bond Max, but I may be hoping for too much! Thanks in advance for your help. Martha Ward, MT (ASCP) QIHC Assistant Manager, Molecular Diagnostics Lab Wake Forest University Baptist Medical Center 336-716-2104 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet - Confidentiality Notice: The following mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. The recipient is responsible to maintain the confidentiality of this information and to use the information only for authorized purposes. If you are not the intended recipient (or authorized to receive information for the intended recipient), you are hereby notified that any review, use, disclosure, distribution, copying, printing, or action taken in reliance on the contents of this e-mail is strictly prohibited. If you have received this communication in error, please notify us immediately by reply e-mail and destroy all copies of the original message. Thank you.
RE: [Histonet] RE: Histonet Digest, Vol 80, Issue 31
Is this what you are looking for? Ng et al Re-hydration Method for Red Blood Cell Lysis Principle: R-ehydration of air dried smears with normal saline for 30 seconds before fixation in alcohol has been found to produce slides of a quality superior or at least equivalent to that of immediate fixed smears. It allowed the cells to adhere better to the slide and lysis of red blood cells provided a clean background for better assessment. Solutions: 1. Normal Saline Sodium Chloride 9g Distilled water 1000ml 2. 95% Ethanol Procedure: 1. After smearing material, air dry slides using a hair dryer. 2. Place slides in Normal Saline for 30sec. 3. Immediately place slides in 95% ethanol and fix for 10 minutes. 4. Stain slides via PAP. Results: RBCs were effectively lysed but epithelial and mesothelial cells were retained. In general, smears showed a decrease in chromaticity of both nuclear and cytoplasmic staining. Cytoplasmic vacuoles were less distinct and epithelial fragments, such as acini, appeared flattened and could be more easily focussed on the same plane. Nuclear and cellular moulding was exaggerated and more appreciable when present. There was a slight enlargement of cells. Degenerated specimens, even in the absence of polymorphs, often resulted in disappointing re-hydrated smears. Cell clusters were easier to see probably because of the higher transparency of the cytoplasm. Reference: Acta Cytolog (1994) 38(1):56-64. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Aazath Raj Sent: Thursday, 12 August 2010 10:48 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] RE: Histonet Digest, Vol 80, Issue 31 Dear Friends, Does anyone have rehydration technique of air dried smear which can be stained for pap. Aazathraj.P Technical Officer Department of Histopathology and Cytology Apollo Hospitals -chennai India ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] PAP
This is our method: 1. Tap water dip gently 5-10 times 2. Harris haematoxylin 5 minutes 3. Wash in running water 4. Differentiate in acid/alcohol one dip 5. Wash in running water 6. Scott's Blueing Solutionone minute 7. Wash in running water. 8. 95% Alcohol dip gently 5-10 times 9. Modified PAP Stain (see below) three minutes 10. Rinse excess dye off in 95% Alcohol 11. Absolute Alcoholdip gently 5-10 times 12. Absolute Alcoholdip gently 5-10 times 13. Xylol dip gently 5-10 times 14. Xylol dip gently 5-10 times 15. Xylol dip gently 5-10 times 16. Mount in D.P.X. Mounting Medium. This modification uses a single solution, omitting the OG6 solution and replacing the light green with the more fade resistant Fast Green FCF. Modified PAP stain Stock solutions: Prepare 10% solutions of each of the stains as follows: 2.5g Eosin Y (CI 45380) in 25ml distilled water 1g Fast Green FCF (CI 42053) in 10ml distilled water Working Solution: Mix (for 400ml stain) 12ml Eosin Y stock 2.5ml Fast Green FCF Stock Make mixture up to 400ml with 95% alcohol. Add: 0.8g Phosphotungstic acid 4 drops saturated lithium carbonate. Mix well. Store solution in dark brown, tightly capped bottle. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Aazath Raj Sent: Thursday, 12 August 2010 10:40 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] PAP Dear friends, does anyone have rapid PAP(cytostain) preparation,for rapid PAP staining. Aazathraj.P Technical Officer Department of Histopathology and Cytology Apollo Hospitals chennai India ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. * ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
