Re: [Histonet] TRAP Stain Help
Travis, Were your samples stored in alcohol? If so, that can kill the TRAP enzyme. Your protocol is similar to ours. Sarah Mack University of Rochester Medical Center Center for Musculoskeletal Research Histology, Biochemistry, and Molecular Imaging Core 601 Elmwood Avenue Box 665 Rochester, NY 14642 (585)-273-3901 From: histonet-requ...@lists.utsouthwestern.edu [histonet-requ...@lists.utsouthwestern.edu] Sent: Tuesday, May 12, 2015 1:00 PM To: histonet@lists.utsouthwestern.edu Subject: Histonet Digest, Vol 138, Issue 14 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu_mailman_listinfo_histonetd=AwICAgc=4sF48jRmVAe_CH-k9mXYXEGfSnM3bY53YSKuLUQRxhAr=ODw50OyFtFWu8REOenc_8wsdRMG_cbkreuWUix7iMVom=DR5hhSofRSHkcr9uY8sQ0Qfr0tmGbuJDCIwuTAylDyIs=ojqKLAQtVhO8ixOrdIb13M0riQkWk2GB5BRQSgbZ3_Qe= or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... Today's Topics: 1. anti-human IgG antibody (Krempley, Amanda L) 2. TRAP Stain Help (Wait, Trevor Jordan) 3. Per Diem Positions at UC San Diego Health System (Paula Sicurello) 4. HE Stainer Question (Paula Sicurello) 5. Re: HE Stainer Question (Roy, Lisa) 6. Re: HE Stainer Question (Sanders, Jeanine (CDC/OID/NCEZID)) 7. Re: HE Stainer Question (Simmons, Christopher) 8. Re: HE Stainer Question (Roy, Lisa) 9. RELIA HOT JOB Alert! Lead Histotech needed for Brand New Lab in Dallas/Ft. Worth. A RELIA EXCLUSIVE!!! (Pam Barker) 10. Re: HE Stainer Question (Joelle Weaver) -- Message: 1 Date: Mon, 11 May 2015 19:52:18 + From: Krempley, Amanda L amanda.kremp...@abbvie.com To: HistoNet histonet@lists.utsouthwestern.edu Subject: [Histonet] anti-human IgG antibody Message-ID: 9667996145fe4392bc5c71b3f3a8a...@usaasecsm048.r0018.collaboration.ecs.hp.com Content-Type: text/plain; charset=utf-8 Hello Histoland, Has anyone used an anti-human IgG antibody that does not cross react with Non-human primates, specifically cynomolgus, for IHC? Could you please share the antibody and protocol that you have had success with? Thank you, Amanda -- Message: 2 Date: Mon, 11 May 2015 20:26:14 + From: Wait, Trevor Jordan wa...@livemail.uthscsa.edu To: Histonet@Lists. Edu histonet@lists.utsouthwestern.edu Subject: [Histonet] TRAP Stain Help Message-ID: 1431375976414.44...@livemail.uthscsa.edu Content-Type: text/plain; charset=iso-8859-1 Hey guys, recently I've used the Sigma Aldrich TRAP Stain Kit in order to stain for Osteoclasts in Formalin Fixed Paraffin Embedded bone tissues that are EDTA decalcified. Unfortunately there was no TRAP stain to be found whatsoever on all slides that I stained. I was hoping that someone with some experience with TRAP stain could really help me out! Here are a couple of reasons that I have wondered as to why the TRAP stain might not be visible 1. Staining too long with Hematoxylin counterstain? I have noticed in several trial runs that sometimes if the Hematoxylin counterstain is too long then this can effect the amount of TRAP stain that shows up with the osteoclasts. Perhaps I'm just over analyzing 2. Perhaps no osteoclasts were present to even staindoes anyone know what the usual ratio of Osteoclasts to Osteocytes are? In the past stains that I have usedwhenever there is an osteoclasts that shows up...it is usually pretty spotty and many times they are in groups together...is this how it normally is? 3. Perhaps the TRAP stain is being washed away through the rinsing with dI or dehydration with ethanol/clearing just before mounting. A protocol that I used before the Sigma Aldrich Kit incorporates incubating the slides in 37 celsius water for 1 hour just prior to allowing the slides to sit in the TRAP stain solution (this is also 37 celsius in the same water bath) and letting it sit for 20 minutes. With this stain...there seems to be a consistent showing of osteoclasts but I'm just not sure if all of the osteoclasts are showing up correctly...that is why I moved to the Sigma Aldrich kit to make sure and the results didn't show ANY osteoclasts there! Anyways...I'm just curious if there are any reasons that the TRAP stain is not showing up...I would appreciate any input! Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate -- Message: 3 Date: Mon, 11 May 2015 13:57:21 -0700 From: Paula Sicurello pat
[Histonet] TRAP Stain Help
If you include a positive slide to your TRAP? It is a must for each run. It could be many things if you didn't have positive slide, will be hard to troubleshot. Also if you see any pink color befor counterstain/coversliping? If do, there may be because of decolored by ethanol/xylene and mounting media. You have to use water based mounting media. I don't use Sigma kit (tried once), but my protocol is more sensitive. We do trap on both paraffin and MMA sections. If you need I can forward you the paper. Dorothy Hu HSDM Message: 2 Date: Mon, 11 May 2015 20:26:14 + From: Wait, Trevor Jordan wa...@livemail.uthscsa.edu To: Histonet@Lists. Edu histonet@lists.utsouthwestern.edu Subject: [Histonet] TRAP Stain Help Message-ID: 1431375976414.44...@livemail.uthscsa.edu Content-Type: text/plain; charset=iso-8859-1 Hey guys, recently I've used the Sigma Aldrich TRAP Stain Kit in order to stain for Osteoclasts in Formalin Fixed Paraffin Embedded bone tissues that are EDTA decalcified. Unfortunately there was no TRAP stain to be found whatsoever on all slides that I stained. I was hoping that someone with some experience with TRAP stain could really help me out! Here are a couple of reasons that I have wondered as to why the TRAP stain might not be visible 1. Staining too long with Hematoxylin counterstain? I have noticed in several trial runs that sometimes if the Hematoxylin counterstain is too long then this can effect the amount of TRAP stain that shows up with the osteoclasts. Perhaps I'm just over analyzing 2. Perhaps no osteoclasts were present to even staindoes anyone know what the usual ratio of Osteoclasts to Osteocytes are? In the past stains that I have usedwhenever there is an osteoclasts that shows up...it is usually pretty spotty and many times they are in groups together...is this how it normally is? 3. Perhaps the TRAP stain is being washed away through the rinsing with dI or dehydration with ethanol/clearing just before mounting. A protocol that I used before the Sigma Aldrich Kit incorporates incubating the slides in 37 celsius water for 1 hour just prior to allowing the slides to sit in the TRAP stain solution (this is also 37 celsius in the same water bath) and letting it sit for 20 minutes. With this stain...there seems to be a consistent showing of osteoclasts but I'm just not sure if all of the osteoclasts are showing up correctly...that is why I moved to the Sigma Aldrich kit to make sure and the results didn't show ANY osteoclasts there! Anyways...I'm just curious if there are any reasons that the TRAP stain is not showing up...I would appreciate any input! Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TRAP Stain Help
Hey guys, recently I've used the Sigma Aldrich TRAP Stain Kit in order to stain for Osteoclasts in Formalin Fixed Paraffin Embedded bone tissues that are EDTA decalcified. Unfortunately there was no TRAP stain to be found whatsoever on all slides that I stained. I was hoping that someone with some experience with TRAP stain could really help me out! Here are a couple of reasons that I have wondered as to why the TRAP stain might not be visible 1. Staining too long with Hematoxylin counterstain? I have noticed in several trial runs that sometimes if the Hematoxylin counterstain is too long then this can effect the amount of TRAP stain that shows up with the osteoclasts. Perhaps I'm just over analyzing 2. Perhaps no osteoclasts were present to even staindoes anyone know what the usual ratio of Osteoclasts to Osteocytes are? In the past stains that I have usedwhenever there is an osteoclasts that shows up...it is usually pretty spotty and many times they are in groups together...is this how it normally is? 3. Perhaps the TRAP stain is being washed away through the rinsing with dI or dehydration with ethanol/clearing just before mounting. A protocol that I used before the Sigma Aldrich Kit incorporates incubating the slides in 37 celsius water for 1 hour just prior to allowing the slides to sit in the TRAP stain solution (this is also 37 celsius in the same water bath) and letting it sit for 20 minutes. With this stain...there seems to be a consistent showing of osteoclasts but I'm just not sure if all of the osteoclasts are showing up correctly...that is why I moved to the Sigma Aldrich kit to make sure and the results didn't show ANY osteoclasts there! Anyways...I'm just curious if there are any reasons that the TRAP stain is not showing up...I would appreciate any input! Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
