[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
