Re: [Histonet] Freezing spray artifact
Erin - Try this trick when using the freezing spray: take a folded kim wipe tissue and fold it again, then again then the long fold is folded twice so you end up with roughly a 1 inch by 1 inch multi layer slide wiper. Run your finger down each crease and after the final fold, give it a little pinch and twist so it holds its shape. You are probably wondering what does this have to do with freezing spray artifact... Dip the square of Kim Wipe in your water bath and press it on the cut surface of your tissue block. It will act as an insulator when you spray the block surface. They are also good for soaking the face of a block and are better than a wad of tissue when wiping slides. JB "Martin, Erin" Sent by: histonet-boun...@lists.utsouthwestern.edu 05/19/2010 12:41 PM To histonet cc Subject [Histonet] Freezing spray artifact Has anyone run into a problem or artifact from freezing spray? I think we may be having a problem with it but I can't find any pictures or descriptions of what it looks like. Thanks in advance, Erin Erin Martin, Histology Supervisor UCSF Department of Dermatopathology 415-353-7248 ___ 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] protein concentration for Cy2
Eric: Are you using Cy2 labeled goat anti-mouse IgG (H&L) from Jackson ImmunoResearch Labs? According to their catalog (product number 115-225-003), the stock is 2.0 mg/ml. At 1:100, your working concentration would be 20 ug/ml. Jim B "Breisch, Eric" Sent by: histonet-boun...@lists.utsouthwestern.edu 04/22/2010 03:51 PM To cc Subject [Histonet] protein concentration for Cy2 This question if for anyone doing C4d immunoflourescence staining. Could someone share what protein concentration the Cy2 labeled anti-mouse IgG is prepared at and what buffer is used for that? Our final dilution of the Cy2 is (1:100 in 1%BSA/PBS) but we don't know what the original protein concentration for the dry Cy2 should be. Thank you for your assistance it is greatly appreciated. Eric A. Breisch ___ 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] CD3 on mouse
Patsy - I agree. LV's rabbit MoAb works well on mouse tissue. They also have a rabbit polyclonal CD20 that works well on mouse. jb "pru...@ihctech.net" Sent by: histonet-boun...@lists.utsouthwestern.edu 03/18/2010 11:58 AM Please respond to pru...@ihctech.net To "Mauger, Joanne" , "Emily Sours" , "histonet@lists.utsouthwestern.edu" cc Subject RE: [Histonet] CD3 on mouse Joanne, the rabbit polyclonal or rabbit monoclonal cd3's work well on mouse tissue, dako has poly and lab vision i believe has a rab mono, it may be poly too, the key is that it is rabbit and not mouse. From: "Mauger, Joanne" Sent: Wednesday, March 17, 2010 1:34 PM To: "Emily Sours" , "histonet@lists.utsouthwestern.edu" Subject: RE: [Histonet] CD3 on mouse Hi All, Does anyone know a CD3 antibody that works well on FFPE mouse tissue? Thanks in advance, Jo Joanne Mauger HT(ASCP)QIHC Children's Hospital of Philadelphia ___ 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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] North Carolina Histology Meeting
To members and friends of the NCSHT: &nbs= p; On behalf of the North Carolina Society of Histopathology Technologist= s (NCSHT) we invite you to our 2010 NCSHT Spring Meeting, April 8 th <= /SUP>- 10 th in Research Triangle Park , NC . <= P class=MsoNormal style="MARGIN: 0in 0.5in 0pt 0in; TEXT-ALIGN: justify= "> Th= e speakers we have selected are very knowledgeable and have a passion for o= ur profession! The meeting = is not only a place to gain a wealth of knowledge but to take the time to v= iew the latest laboratory products and instrumentation on the market today.= So we hope you make time t= o visit the exhibits. Mark = your calendars for the NCSHT Spring Meeting, April 8 th - = 10 th . &nbs= p; We look forward to seeing you in RTP! Warmest Regards, The Officers of NCSHT <= P class=MsoNormal style="MARGIN: 0in 0in 0pt"> Please contact Lisa Gates or Jim= Burchette for an electronic copy of the program. &n= bsp; President: == &nb=sp; &nb= sp;Vice President: = ;&n bsp; = ;&n= bsp; L= isa Gates &nbs= p;&= nbsp; &nbs p; &= nbsp; Jim Burchette [1]lisa.d.ga...@gsk.com &nb= sp; [2]burch...@mc.duke.edu References 1. file://localhost/tmp/3D"mai 2. ="mailto:burch...@mc.duke.edu"; ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Procedure for manual C4D immunofluorescence staining
Frozen sections cut at 3-4 microns. Air dry 30 minutes. Acetone fix for 5 minutes (room temp acetone), air dry slides PBS wash x2 C4d (diluted 1:500 in 1% BSA/PBS) incubated 1 hour. Mouse monoclonal C4d is from AbDirect/Serotec PBS rinse, wash x2 Cy2 labeled goat anti-mouse IgG (diluted 1:100 in 1% BSA/PBS), incubated 30 minutes (Cy2 GAR from Jackson ImmunoResearch Labs) PBS rinse, wash x2 Coverslip with fluorescence mounting media Happy viewing I run this protocol everyday! Jim Burchette, HT(ASCP) QIHC "A simple histotech from a little country hospital in North Carolina" "Foshey, Annette" Sent by: histonet-boun...@lists.utsouthwestern.edu 10/15/2009 04:56 PM To " (histonet@lists.utsouthwestern.edu)" cc Subject [Histonet] Procedure for manual C4D immunofluorescence staining Histonetters, Does anyone have a procedure they could share for manual C4D immunofluorescence staining? Thanks in advance Annette Foshey, HT (ASCP) Charge Tech in Histology Children's Hospital of Wisconsin 414-266-6580Fax 414-266-2779 afos...@chw.org ___ 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] DIF tissue in GLUT
Dear Anne, I assume the glutaraldehyde tissue has been processed and embedded in paraffin. Following removal of paraffin and any pretreatment to unmask the antigen, you can block autofluorescence or change the color by treating the sections with 0.05% sodium borohydride prepared in PBS for 30 minutes. After the borohydride treatment, carefully wash the slides with multiple changes of PBS for thirty minutes. I would also use a good quality silane treated slide for maximum tissue adhesion. A comment of caution: sodium borohydride is poisonous. Be extremely careful with the powder. Reference for NaBH4: Embedment in Glycol Methacrylate at Low Temperature Allows Immunofluorescent Localization of a Labile Tissue Protein. JA Carnegie, ME McCully and HA Robertson, Journal of Histochemistry and Cytochemistry, Vol. 28, No. 4, pp 308-310, 1980 Methods in Cell Biology, by Leslie Wilson, page 117, section VI, Use of Glutaraldehyde in Immunofluorescence Microscopy. Academic Press 1982 I used this method in a double label fluorescence project a few years ago with excellent success. 23. Endothelial Metaplasia in the Iridocorneal Endothelial (ICE) Syndrome. DN Howell, T Damms, JL Burchette, JR Ross, WR Green. Investigative Ophthalmology and Visual Science, Vol. 38, No. 9, pages 1896-1901, August 1997. Best Regards, Jim Jim Burchette, HT(ASCP) QIHC "A simple histotech from a little country hospital in North Carolina" Anne van Binsbergen Sent by: histonet-boun...@lists.utsouthwestern.edu 08/27/2009 06:27 AM To John Kiernan , "histonet@lists.utsouthwestern.edu" cc Subject [Histonet] DIF tissue in GLUT Dear John and other knowledgeable Histonetters and Gurus of the trade Renal bx tissue core has been placed in Gluteraldehyde/Trumps EM fixative by mistake (we all make mistakes) - we now need to do DIF Does anyone out there have a method for this? -- Anne van Binsbergen (Hope) Abu Dhabi UAE ___ 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
[Histonet] Job Opening in North Carolina
The Immunopathology Laboratory at Duke University Medical Center in Durham NC has a technician/technologist position available. Please contact me for further information. Jim Burchette burch...@mc.duke.edu 919.681.3973 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] whipf's polychrome procedure
Here is the polychrome formula I use. Jim Polychrome Methylene Blue Stain Tissue and Fixation Frozen sections and formalin-fixed, paraffin embedded Objective Rapid differential staining of tissue sections. Metachromatic stain. Reagents Stock solutions: A. 12% aqueous potassium carbonate, 100 ml. B. 1% aqueous methylene blue, 4000 ml. C. 10% aqueous glacial acetic acid, 50 ml. Staining solutions: 1. Mix 100 ml of A with 5000 ml of B. 2. Gently boil for 60 minutes. Cool to room temperature. 3. Add 50 ml of C. 4. Store in a loosely capped bottle for further oxidation. Procedure 1. Dip frozen sections in stain for 5 seconds. 2. Rinse excess stain from slide. 3. Mount in water with a cover glass if desired. Results Nuclei blue, cytoplasm, muscle, connective tissue, pink to purple. Note: The sections are not permanent. Solution works best after aging for at least 12 months, recognizable by a rich tinctoral quality. Increase stock solution volumes to make a larger quantity of working stain. Filter before use. Reference: Terry, BT, J. Lab and Clinical Medicine, Vol. 13, page 550, 1928. "Woodward, Denise" Sent by: histonet-boun...@lists.utsouthwestern.edu 08/11/2009 11:23 AM To "histonet@lists.utsouthwestern.edu" cc Subject [Histonet] whipf's polychrome procedure Hello all, One of our veterinary pathology residents from France is looking for a staining procedure for "Whipf's Polychrome". Anyone have it and willing to share? Thanks, Denise Long Woodward University of Connecticut Connecticut Veterinary Medical Diagnostic Laboratory Dept. of Pathobiology and Veterinary Sciences ___ 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] Re: Knife strop photo?
I sent her a digital image of one we have here at our little hospital. Jim Burchette, HT(ASCP) QIHC "A simple histotech from a little country hospital in North Carolina" Robert Richmond Sent by: histonet-boun...@lists.utsouthwestern.edu 06/11/2009 01:40 PM To histonet@lists.utsouthwestern.edu cc Subject [Histonet] Re: Knife strop photo? Esther Peters requests a picture of the stropping and honing technique once used (long before this geezer's time) to sharpen microtome blades. Here's a scan, with captions, on my Flickr Web site at http://www.flickr.com/photos/bobrichmond/3616614043/ This is probably out of copyright. I can supply a higher definition scan if you need one for printing purposes. Bob Richmond Samurai Pathologist Knoxville TN ___ 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] immunohistochemistry
I second Mr. Troutman's recommendation using silane slides, = but air dry overnight. Once the slides have been baked and paraffin re= moved, place the slides in citrate buffer and heat them in a closed plastic coplin jar for 12 hours at 75 degrees C. Some epitopes can be retrieved wi= th this retrieval process. It's worth a try. Jim Burchette, H= T(ASCP) QIHC "A simple histotech from a little country hospital in North= Carolina" ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: [IHCRG] CMV antibody
Dear Jean, I am very fond of Dako's M0854 and find I can use it at 1:400-1:500 when pepsin is used as a pretreatment proteolytic enzyme as compared to HIER pretreatment with a citrate buffer. CMV is run everyday in my lab. Jim Burchette "Taylor, Jean" Sent by: ih...@googlegroups.com 05/20/2009 03:06 PM Please respond to jtay...@meriter.com To "'ih...@googlegroups.com'" , "'histonet@lists.utsouthwestern.edu'" cc Subject [IHCRG] CMV antibody Hi Everyone, I’m wondering what antibody clone labs are using to detect Cytomegalovirus by IHC. Thank you! Jean Taylor, HT(ASCP) QIHC IHC Tech. Meriter Health Services Madison, WI 53715 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] HTLV-1 IHC
Is anyone performing IHC on FFPE human tissue for HTLV-1? Many Thanks, Jim ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] NC Society for Histopathology Technologists 2009 Meeting
The North Carolina Society for Histopathology Technologists invites you to our 2009 meeting to be held May first and second at the DoubleTree Biltmore Hotel in beautiful Asheville North Carolina. Call the DoubleTree Biltmore Hotel at 1-800-222-8733 for special room rates and reservations or visit them on line at www.biltmoreasheville.doubletree.com. Make sure you mention NCSHT. Our program is nearly finished and will be posted on the NSH web site next month. We look forward to seeing you in Asheville The officers and board members of NCSHT ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] NGF p75
Leica Microsystems # NCL-NGFR "Martin, Erin" <[EMAIL PROTECTED]> Sent by: [EMAIL PROTECTED] 11/12/2008 12:06 PM To histonet cc Subject [Histonet] NGF p75 Good morning, Does anyone know of a source for antibody NGF p75 other than Chemicon/Millipore? The last few lots we have gotten from them have been dramatically weaker so I'd like to find a new vendor. Thanks! Erin ___ 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] Pepsin
Gene: Dako and Biocare both sell the stable pepsin solution. It is easy to prepare if you want to make it yourself. I use this pepsin everyday. Here is the recipe that Dr. Brigati gave me. Best Regards, Jim Burchette Brigati’s 0.25% Stable Pepsin Solution Reagents Pepsin, #P7012, Sigma Chemical Co. ** 10X Automation Buffer, #BMM30, Fisher Scientific ** Preparation 40 ml 10X Automation Buffer 356 ml distilled water Adjust mixture of Automation Buffer and water to pH 2.0. Add 1.0 gram of P7012 pepsin (it is important to add the pepsin after the pH adjustment). Place container on a stir plate with gentle stirring allowing the pepsin to dissolve. The solution is sometimes cloudy at first, but will become clear after sitting overnight. Store at 4 C. Apply at room temperature Digest formalin-fixed, paraffin embedded tissue sections for 13-15 minutes at 37-40. Wash with IHC procedural buffer or DI water to stop enzyme digestion and to neutralize the pH. ** The Biomeda TBS automation buffer is no longer available. I now use the Automation Wash TBS Buffer from Biocare Medical. It is a 20X concentrate. Adjust the volume of water and buffer accordingly. You could also use 396 ml of 1X TBS. Dr. Brigati passed away a year ago. He was a great pathologist, mentor and friend. [EMAIL PROTECTED] Sent by: [EMAIL PROTECTED] 10/10/2008 09:05 AM To Histonet@lists.utsouthwestern.edu cc Subject [Histonet] Pepsin Does anyone have a catalogue # for the Pepsin used in Brigati's stable pepsin? Thanks, Gene Cleveland Clinic ___ 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] SV40 HRP protocol HELP
Dear Anne: I use the SV40 (catalog # DP02) from Oncogene, a division of Calbiochem. A dilution of 1:500 consistently works well from lot to lot. For manual or autostainer platforms, pretreatment is with Dako's Target Retrieval pH 6.1 solution for 20 minutes in a 100 C water bath. Following a one hour primary incubation, the bound antibody is detected with HRP labeled Dako Envision Plus (30 minute incubation). I like using an enhanced DAB, such as Dako's DAB+. I find this SV40 also works well on the Leica Bond Max slide stainer. The same 1:500 dilution is used and the Bond pretreatment is programmed for 20 minutes with HIER solution #1 (aka ER1). Other than the pretreatment programming, their standard incubation schedule for all of the subsequent steps works well. Best Regards Jim Burchette http://www.emdbiosciences.com/Products/ProductDisplay.asp?catno=DP02&; Catalog # DP02 Host: Mouse Isotype: IgG2a Immunogen: purified SV40 large T-antigen Epitope: Within amino acids 83-128 Form: Liquid Formulation: In 50 mM sodium phosphate buffer, 0.2% gelatin, pH 7.5. Preservative: ≤0.1% sodium azide Positive Control: SV80 or SV-T2 cells Negative Control: 3T3 cells Comments: Recognizes ~94 kDa SV40 large T antigen in SV80 and SV-T2 cells. Ref.: DeCaprio, J.A., et al. 1988. Cell 54, 275. Whyte, P., et al. 1988. Nature 334, 124. Mitchell, P.J., et al. 1987. Cell 50, 847. Dixon, R.A.F. and Nathans, D., 1985. J. Virol. 53, 1001. Simanis, V. and Lane, D.P. 1985. Virol. 144, 88. Mann, R.S. and Carroll, R.B. 1984. Virology 138, 379. Sarnow, P., et al. 1982. Cell 28, 387. Crawford, L.V., et al. 1981. Proc. Natl. Acad. Sci. USA 78, 41. Lane, D.P. and Crawford, L.V. 1979. Nature 278, 261. Carroll, R.B., et al. 1974. Proc. Natl. Acad. Sci. USA 71, 3754. Tooze, J. 1973. Cold Spring Harbor, New York. Tegtmeyer, P. 1972. J. Virol. 10, 591. "Anne van Binsbergen" <[EMAIL PROTECTED]> Sent by: [EMAIL PROTECTED] 10/09/2008 08:05 AM To histonet , [EMAIL PROTECTED] cc Subject [Histonet] SV40 HRP protocol HELP Hello Histonetters please could someone out there (anyone) assist with a working protocol for Immuno SV40 (HRP) TIA abudhabiannie ___ 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
