[Histonet] Double stain IHC question
This is probably most easily done using immunofluorescence since it works well on frozen tissue. You could use a streptavidin linked FITC tag for the human biotinylated Ab, and then use a red tagged anti IgG directed against the species of the other antibody. (hoescht reagent will stain the nuclei) If however you want to use immunohistochemistry you can use a strepatvidin linked alkaline phosphatase with BCIP/NBT (blue chomogen) for the biotinylated Ab and a polymer based peroxidase secondary for the other antibody and visualise using DAB. For nuclear staining you can use nuclear fast red. I have used this method recently to stain cytospots successfully for similar macrophage markers (inos and cd68). Hope this helps. regards steve weston lab manager Breathe-Well CRE UTAS-SOM University of Tasmania Electronic Communications Policy (December, 2014). This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re best glass coverslipper
I've been using the Dako coverslipper for over three years now. Once i had got used to ensuring the sliding mechanism was always kept mountant free and regularly changed the xylene in the u tube we have had very few problems with this machine. Its footprint is without question the smallest coverslipper i have seen and generally with good maintenance (yearly PM) it functions very well. Occasionally we get small bubbles under the coverslips but they are easily removed. Highly recommend looking at one, also the price is very good. regards steve weston lab manager Breathe-Well CRE UTAS-SOM University of Tasmania Electronic Communications Policy (December, 2014). This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] H and E Staining in IHC
Hi Marcus, As stated previously by others there should be no problem putting the Eosin into your sections but I would suggest that you do not leave out the endogenous quenching so you can show your red blood cells. The reason being that there are many other cells that may then show up as positive. Instead I suggest using an Eosin/Phloxine mixture which nicely highlights the red cells differently to the rest of the general matrix material. regards Steve Weston Breathe-Well CRE Lab Manager 0408990859 University of Tasmania Electronic Communications Policy (December, 2014). This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Help with causes or theory behind failure of nuclear counterstaining after IHC! (Maria Mejia)
Maria, This may sound simplistic but I find that if I have problems such as this the first thing I do is try a new batch of the staining reagents. If you haven't had problems before then something must have changed either in your protocols or your reagents. It could be that your heat retrieval reagents are too old or contain detergents that remove some of the proteins you are looking for. Some of the proprietary heat retrieval reagents that allow you to heat retrieve without having to dewax by going through xylene have been shown to change the nuclear staining pattern and create what appear to be nuclei that are full of vacuoles. Also if the celloidion is not completely removed during your staining it may be stopping any of the higher molecular weight stains from penetrating the cells. Try leaving your sections in acetone for a number of changes to ensure full removal of the celloidion. Regards Steve Weston University of Tasmania Breathe-Well CRE Lab Manager 0408990859 University of Tasmania Electronic Communications Policy (December, 2014). This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re automatic cover slippers
We use the Dako cover slipper and provided the moving arm is kept free from dried mounting medium we have found this to be a terrific machine. It also has a really small footprint and so fits nicely into our fume hood. Can do about 400 slides an hour and produces very few bubbles. Steve Weston Breathe-Well CRE Lab Manager 0408990859 University of Tasmania Electronic Communications Policy (December, 2014). This email is confidential, and is for the intended recipient only. Access, disclosure, copying, distribution, or reliance on any of it by anyone outside the intended recipient organisation is prohibited and may be a criminal offence. Please delete if obtained in error and email confirmation to the sender. The views expressed in this email are not necessarily the views of the University of Tasmania, unless clearly intended otherwise. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] CD4 1F6 clone
I also tried it without H2O2 with no result either. Anyone else had these problems with this clone ??? Interestingly in the past I used the duo pack of CD4/8 that had the 1F6 for cd4 and the 4B11 clone of cd8 and they both worked. May have to try the 4b12 clone for cd4. regards Steve Weston Message: 10 Date: Sat, 30 Aug 2014 15:44:09 +0530 From: Neeraj Singh neerajbioxf...@gmail.commailto:neerajbioxf...@gmail.com Subject: Re: [Histonet] Re cd4 1F6 clone To: Steven Weston steven.wes...@utas.edu.aumailto:steven.wes...@utas.edu.au Cc: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Message-ID: 1663fc3e-e152-411f-b553-83d3d426a...@gmail.commailto:1663fc3e-e152-411f-b553-83d3d426a...@gmail.com Content-Type: text/plain; charset=us-ascii Try with no H2O2 at all for this clone. it will hill you. Thanks Neeraj On Aug 30, 2014, at 1:40 PM, Steven Weston steven.wes...@utas.edu.aumailto:steven.wes...@utas.edu.au wrote: Hi Histonetters Has anyone else had difficulty getting this clone to work? cd4 1F6 from leica microsystems (formerly novocastra). I've tried just about every trick in my book and still no staining. Particularly i made sure i did the peroxidase block prior to HIER (i even tried using it after the primary) and have tried low, high and middle pH buffers with antibody concentrations from 1/10-1/40 for 60 and 90 mins and use envision plus detection with DAB plus. I've had two batches of this over the last ten years and neither has worked. Anyone else with the same experience ? regards steve weston lab manager Breathe-Well CRE UTAS-SOM Steve Weston Lab Manager Breathe-Well Centre of Research Excellence for Chronic Respiratory Disease. UTAS-SOM 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re cd4 1F6 clone
Hi Histonetters Has anyone else had difficulty getting this clone to work? cd4 1F6 from leica microsystems (formerly novocastra). I've tried just about every trick in my book and still no staining. Particularly i made sure i did the peroxidase block prior to HIER (i even tried using it after the primary) and have tried low, high and middle pH buffers with antibody concentrations from 1/10-1/40 for 60 and 90 mins and use envision plus detection with DAB plus. I've had two batches of this over the last ten years and neither has worked. Anyone else with the same experience ? regards steve weston lab manager Breathe-Well CRE UTAS-SOM ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re Electron microscope choices ?
Hi all, I'm still looking for ideas about the best types of TEM around at the moment. If you have recently purchased one or have been a regular user of a late model digital TEM please share your experience with me. regards Steve Weston Lab Manager Breathe-Well Centre of Research Excellence for Chronic Respiratory Disease. UTAS-SOM 0408990859 62264871 Steve Weston Lab Manager Breathe-Well Centre of Research Excellence for Chronic Respiratory Disease. UTAS-SOM 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] TEM choices
HI there fellow histonetters, We are in the fortunate position of being able to purchase a new TEM and was wondering what you all think of the present choices on market. We wish to get a digital machine to replace an ageing Joelle. Any feedback from users about recent purchases would be welcome, including ease of use, maintenance, support and reliability. regards Steve Weston Lab Manager Breathe-Well Centre of Research Excellence for Chronic Respiratory Disease. UTAS-SOM 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re wrinkles in section
Hi Margaret, One possible solution to the wrinkling problem you have is to float your twenty microns onto the slide. I do this regularly with mouse or rat brain by cutting the section and putting it into culture wells with buffer solution. Then once I have completed my sectioning I lift the individual sections using a hockey stick made from a bent glass pasteur pipette (although a good fine paintbrush works as well) and dip them into a 30% ethanol solution briefly and then onto a distilled water bath. When you lower the section onto the water bath (at room temp) it should spread due to surface tension changes, and then you can pick it up onto a charged slide as you would a paraffin section. You then allow the section to dry on the slide. This also removes the OCT and gives you nice sections on clean slide that, once dried, stain well. (I usually dry in a 37 degree incubator overnight). Hope this helps Steve Weston Lab Manager Breathe-Well Centre of Research Excellence for Chronic Respiratory Disease. UTAS-SOM 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re 2050 microtome
This could be as simple as moving the specimen holder forward. It may be racked right into the microtome and therefore show as stopped. Try moving the holder forward and see if the stopped light goes off. Regards steve weston lab manager Breathe-Well CRE UTAS-SOM Message: 9 Date: Sat, 15 Jun 2013 00:07:51 -0300 From: C.D.G. late...@montevideo.com.uy Subject: [Histonet] manual setup for Reichert-Jung 2050 needed To: histonet@lists.utsouthwestern.edu Cc: histonet@lists.utsouthwestern.edu Message-ID: 201306150007510574.0040b...@smtp.montevideo.com.uy Content-Type: text/plain; charset=ISO-8859-1 Hi all: i received a Reichert-Jung microtome 2050 model. I need instructions for its operation. The electronic panel displays stop illuminated and I dont know how to continue, as other buttons seems not to operate. Any help of people who has worked or know to operate this motorized microtome will be appreciated. My kind regards, Carlos Defeo Histotechnologist ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re problem with DAB staining
Another suggestion is that your sections may have dried out sometime after the antibodies have been added and before you put on the DAB. regards steve weston lab manager resp research group MRI ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re vacuoles in nuclei of PT Link treated tissue
Hi everyone, I have recently encountered an unusual result in our immunostaining. We have been bvery successfully using Dako's PT link with the High PH soln to dewax and heat retrieve some of our immuno samples. In conjunction with my colleagues from the local Hospital Anat Path dept we noticed a strange but prominent vacuolisation showing in the Nuclei (and less so in cytoplasm) in our treated tissue after staining with Haematoxylin. Here is the interesting thing, when we manually dewax the same type of sample and then through the high PH, PT link the vacuoles no longer appear. This was noticed in both laboratories. the first thought was that it had something to do with the wax. But we both use different waxes (precision cut and paraplast). Clearly it is something to do with the wax removal process but coming up with an explanation is challenging. Any ideas? Has anyone else noticed this phenomenon ? regards steve weston lab manager resp research group MRI ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re molds
Topic: cleaning holds From: histonet-boun...@lists.utsouthwestern.edumailto:histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of O'Donnell, Bill Sent: Monday, October 08, 2012 2:32 PM To: histonet@lists.utsouthwestern.edumailto:histonet@lists.utsouthwestern.edu Subject: [Histonet] Metal molds OK folks, I know I should be smarter than this and I haven't seen discussion on it lately Are people cleaning their metal embedding molds after evey embedding session? If not, how often do you clean them? Do you clean them at all? If you clean them, how do you do it? Thanks Bill William (Bill) O'Donnell, HT (ASCP) QIHC Senior Histologist Good Samaritan Hospital 10 East 31st Street Kearney, NE 68847 SERENITY is not freedom from the storm, but peace amid the storm. Cultivate it in PRAYER! We clean our holds by boiling them in a metal bowl in water with dishwashing liquid and occasional agitation on a hotplate in the lab. Let them come to boiling and stir a few times to release any wax, then allow the whole thing to cool. The bulk of the wax should form a skin on the top of the water which can be removed and thrown away in the bin. The holds can then be rinsed well in hot tap water to remove any residual wax and then dried in a drying oven. Steve Weston Lab Manager Centre of Research Excellence for Chronic Respiratory Disease. Menzies Research Institute 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re cutting at 50 microns
The neurobiologists in our organisation regularly cut 50 micron sections on our leica cryotome. It is usually easy to do so particularly with brain tissue that has been prepared by fixing in parafomaldehyde and then incubating for a couple of days in 30% sucrose in buffer. The tissue can then be frozen directly in OCT (or similar) the cryostat using the fast freezing peizo area. If the cryostat doesn't adjust to 50 microns then the trick is to set it at 25microns and move the block down to just above the knife then back up again this will move the block forward 25 microns and then as you bring it down again it will move the second 25 microns giving you sections at 50. For brain tissue it should be cut at between –15 and –12 degrees C. Hope that all makes sense. Steve Weston Lab Manager Centre of Research Excellence for Chronic Respiratory Disease. Menzies Research Institute 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] re coverslippers
Hi everyone, Just considering my options for the purchase of a new cover slipper and wanted some feedback on + and – of different types. I am mostly considering the new Dako one but would be interested to hear about peoples experience with the leica and others. regards Steve Weston Lab Manager Centre of Research Excellence for Chronic Respiratory Disease. Menzies Research Institute 0408990859 62264871 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re tunel staining
I recently had an enquiry from one of our post docs who was looking at TUNEL staining and came across this reference in biochemica no 4 (1997)titled “Fixation of Tissue Sections for TUNEL Combined with Staining for Thymic Epithelial Cell Marker” You should be able to google it and find the article. Basically it said that after fixation with PFA to improve staining you need to treat with triton and sodium citrate in the cold to expose the antigen again. Below is the suggested protocol for thin cryostat sections. I’m sure it could be modified for thick sections. 1. Cut 4 μm thin cryosections and mount on polylysine-coated glass slides. 2. Air dry overnight at room temperature and freeze foil- wrapped slides at –20°C until use. 3. Apply frozen glass slides directly into a container with 1% buffered paraformaldehyde for 30 min at RT. 4. Rinse swiftly in PBS and immerse in a solution of 1% Triton X100 (v/v) and 1% sodium citrate (w/v) for 2 min at 4°C. 5. Wash in PBS and incubate with 50 μl TUNEL reaction mixture (TdT solution with dUTP-FITC solution, 1+9). Convert to enzyme label if desired. 6. Wash with PBS and block with dilution of adequate normal serum. 10. Proceed with desired immunohistochemical labeling. Regards Steve Weston Senior technical officer Menzies Research Institute Hobart Tasmania ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re water problem
We had this problem when we started using APTS coated slides and left out adhesive from the water bath. A simple solution I have found is to add a single drop of triton x100 (or similar detergent) to my full water bath and mix before heating the water. This reduces the surface tension of the water and allows it to run off the slide. Regards Steve weston Menzies Research Institute Hobart Tasmania, Australia ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
