Re: [Histonet] nissl after permount?
Thank you for the excellent information. -Teresa On Mon, Dec 5, 2011 at 1:03 AM, John Kiernan jkier...@uwo.ca wrote: The usual approach would be to do Nissl stains on nearby (ideally adjacent) sections to those immunostained for the immediate early gene products. If you have no unstained slides (from bad planning), you will need to remove the coverslips from some of your slides, rehydrate and then do your Nissl stain. If the immunohistochemical product was oxidized DAB (brown), this will remain in place, and your sections will also have nuclear chromatin and nucleolar and cytoplasmic rRNA coloured blue, violet or red according to your choice of Nissl stain. Removing coverslips, extracting resinous mountant, rehydration and restaining is a tedious and time consuming job (days to a few weeks), especially if the slides are bearing thick sections (50-200um) or whole-mounts. As a graduate student, you should have an experienced faculty member to advise you. If your boss told you to ask on the Internet, he isn't earning his salary. There are many experienced histotechnologists at the University of California at Davis. Histotechs love to pass on their experience, learning and practical advice. You should still chase up your academic supervisor, who is paid from your tuition fees. John Kiernan Anatomy, UWO London, Canada = = = On 04/12/11, *Teresa Iglesias *tligles...@ucdavis.edu wrote: Hi all, I'm new to this so this may be a very dumb question but here goes. If you have already stained for IEGs (Zenk and cFos protein) in brain tissue and adhered coverslips with permount to the slides is it possible to then stain with nissl (after removing the covers, of course)? I'm having a hard time locating certain nuclei now (they were obvious before staining for IEGs and mounting) so I'm looking for a way to corroborate that I am looking in the right areas. As an alternative, I can stain new sections with nissl that have been kept in cryoprotectant and are free of IEG staining. IF I take this route, would I have to stain a replicate set of sections for ALL individuals (birds) so that I compare each IEG stained section to a nissl section that came from the same bird? In other words, I have 100 slides with 3+ sections per slide with IEG staining, will I need 100 slides with the same sections stained for nissl? Thanks for the help!! -Teresa -- __ Teresa Iglesias Graduate Group in Animal Behavior Department of Evolution and Ecology University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- __ Teresa Iglesias Graduate Group in Animal Behavior Department of Evolution and Ecology University of California-Davis One Shields Avenue 2320 Storer Hall Davis, CA 95616 Office: 530-754-7837 Fax: 530-752-1449 tligles...@ucdavis.edu __ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] nissl after permount?
Hi all, I'm new to this so this may be a very dumb question but here goes. If you have already stained for IEGs (Zenk and cFos protein) in brain tissue and adhered coverslips with permount to the slides is it possible to then stain with nissl (after removing the covers, of course)? I'm having a hard time locating certain nuclei now (they were obvious before staining for IEGs and mounting) so I'm looking for a way to corroborate that I am looking in the right areas. As an alternative, I can stain new sections with nissl that have been kept in cryoprotectant and are free of IEG staining. IF I take this route, would I have to stain a replicate set of sections for ALL individuals (birds) so that I compare each IEG stained section to a nissl section that came from the same bird? In other words, I have 100 slides with 3+ sections per slide with IEG staining, will I need 100 slides with the same sections stained for nissl? Thanks for the help!! -Teresa -- __ Teresa Iglesias Graduate Group in Animal Behavior Department of Evolution and Ecology University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Vectastain elite ABC kit- multiple dips ok?
Hi all, I have three 24-well plates with three brain sections in each well. For IHC, do I have to prepare three different/fresh ABC dips for each 24-well screen plate or can I incubate (30min) each successive screen plate into the already used dip of ABC? Do I have to leave it in longer to allow adequate binding? I know you can do this with DAB but you have a visual indicator that you've left it in long enough in that case. Any experience with this? Thanks, -Teresa -- __ Teresa Iglesias Graduate Group in Animal Behavior Department of Evolution and Ecology University of California-Davis __ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: transporting sectioned tissues
Is there any way to relax curled brain sections? They've been in cryoprotectant and I tried sitting them in PBS for a while. They're to be used in an IHC- can they be rescued? Thanks, -- Teresa Iglesias Graduate Group in Animal Behavior University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Help! Curled cryosections
Is there any way to relax curled brain sections? They've been in cryoprotectant and I tried sitting them in PBS for a while. They're to be used in an IHC- can they be rescued? Thanks, -- Teresa Iglesias Graduate Group in Animal Behavior University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Help! Curled cryosections
Thanks, Montina The sections are 40um and I had no trouble with all my other brains (sectioned myself). I had help with these last two and they are all curled. I think they just got sectioned too fast or something. I'll try the shaker and see if it helps. Thanks! -Teresa On Wed, Sep 15, 2010 at 8:47 PM, Montina Van Meter montina.vanme...@pbrc.edu wrote: Teresa, I would put the sections through several 5-10 min. washes on a shaker table. It is very important to make sure you have all of the cryoprotectant rinsed out of the tissue or it will inhibit IHC staining. How thick are the sections? Were they cut on a cryostat or freezing microtome? I routinely cut rat brain at 40um and don't have any curling issues. That sometimes occurs when the knife has come through the section of brain too rapidly. A slow and steady motion is needed when cutting frozens. Good luck! Tina Van Meter Sent from my iPhone On Sep 15, 2010, at 9:37 PM, Teresa Iglesias tligles...@ucdavis.edu wrote: -- __ Teresa Iglesias Graduate Group in Animal Behavior Department of Evolution and Ecology University of California-Davis One Shields Avenue 2320 Storer Hall Davis, CA 95616 Office: 530-754-7837 Fax: 530-752-1449 tligles...@ucdavis.edu __ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] delaying Hematoxylin Eosin staining?
I'm a guest in a lab with very little freezer/fridge space so I'm wondering if I can mount fixed-frozen testes tissue onto slides, and once dry, store in a slide box at room temperature for a few weeks until all my slides are ready for staining. Tissues are fixed in 10% formalin then sunk in 20% sucrose. I'll embed in OCT (optimal cutting temperature compound) before cryosectioning. I will mount them after a rinse in PBS (phosphate buffered solution). I worry about the tissues over drying since they are not embedded in paraffin. Does anyone have any experience with this? Thanks! -- Teresa Iglesias Graduate Group in Animal Behavior University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] transporting sectioned tissues
I have brain tissue sectioned and kept in cryopreservative in -20C in 24-well plates but need to take it all to another state when I move. I'm considering using 50ml falcon brand tubes topped to the brim with cryoprotectant to transport them in a checked bag. Is it necessary to keep these vials cold, such as with dry-ice? How have others transported sectioned tissues before? Thanks, -- Teresa Iglesias Graduate Group in Animal Behavior University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] delaying Hematoxylin Eosin staining?
Thanks, Andrea! Is it imperative to rehydrate in the fixative or can I just go straight to rehydration in water? Is rehydrating in fixative because of the long dry-time because I don't see it in any protocols? Thanks again! --Teresa University of CA, Davis On Tue, Aug 31, 2010 at 8:30 PM, andreahoo...@rocketmail.com wrote: H+E staining should be unaffected by storage of frozen sections at RT. I have done this for at least one week and its not a problem. It is IHC or enzymatic stains which could pose a problem - depending on fixative etc. I do not suggest you rinse in PBS before airdrying, however. I take off cryostat directly onto slide and air dry. Air dry cryosections in OCT and leave at RT. When ready to stain, just rehydrate in your fixative for 5-10 min then PBS or water and proceed. Sent from my Verizon Wireless BlackBerry -Original Message- From: Teresa Iglesias tligles...@ucdavis.edu Sender: histonet-boun...@lists.utsouthwestern.edu Date: Tue, 31 Aug 2010 19:38:32 To: histonet@lists.utsouthwestern.edu Subject: [Histonet] delaying Hematoxylin Eosin staining? I'm a guest in a lab with very little freezer/fridge space so I'm wondering if I can mount fixed-frozen testes tissue onto slides, and once dry, store in a slide box at room temperature for a few weeks until all my slides are ready for staining. Tissues are fixed in 10% formalin then sunk in 20% sucrose. I'll embed in OCT (optimal cutting temperature compound) before cryosectioning. I will mount them after a rinse in PBS (phosphate buffered solution). I worry about the tissues over drying since they are not embedded in paraffin. Does anyone have any experience with this? Thanks! -- Teresa Iglesias Graduate Group in Animal Behavior University of California-Davis ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- __ Teresa Iglesias Graduate Group in Animal Behavior Department of Evolution and Ecology University of California-Davis One Shields Avenue 2320 Storer Hall Davis, CA 95616 Office: 530-754-7837 Fax: 530-752-1449 tligles...@ucdavis.edu __ ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
