[Histonet] Problems using tomato lectin
Hi all, I've just purchased Texas-Red conjugated tomato lectin from Vector and have been trying it out on some frozen mouse liver sections but cant see anything. Protocol - fresh frozen sections 8um cut and air dried overnight. Fixed (10min) acetone, washed PBS, tomato lectin applied (I've done serial dilutions from 1/100 - 1/5000 in PBS/0.05% Tween) 1hr at room temp, wash PBS, mount Vectashield + dapi. Any suggestions? Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Mouse abs on mouse tissue method
I've been trying out the method from IHC world to use mouse primary plus anti-mouse secondary abs on mouse tissue. Protocol; Fresh frozen sections (mouse spleen) cut 8um and air dried Fixed acetone 10min Wash PBS Block with 2.5% normal goat serum in PBS/Tw, 30min Block with unconjugated Fab fragment goat anti-mouse IgG (Jackson Immuno, diluted 1/10 in PBS/Tw), 2hrs room temp Wash PBS/Tw Seconary ab - DyLight488 conjugated Fab fragment goat anti-mouse IgG (Jackson Immuno, 1/500 PBS/Tw), 20min room temp Wash PBS/Tw Mount Blocking with unconjugated Fab did decrease background if I used an IgG goat anti-mouse secondary but the background was actually increased when I used the Fab goat anti-mouse!! Any suggestions? Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Murine Intestine Protocols (Lyn Stadler)
Are you doing paraffin or frozen? We do quite a lot of frozen colon / ileum so I can give you our protocols if you like. Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Frozen sectioning human spleen
I've been trying to get some frozen sections of human spleen. Usually all the work I do is with mouse tissue and I take the tissues myself and immediately freeze in OCT in isopentane/dry ice. The problem with the human samples is that I have no control over how they are frozen! I have one sample from our path dept which was just put directly in the -80oC freezer the other sample I have is from LN2 storage (I presume it was snap frozen in LN2 after removal). Anyway the sections from both samples are awful! They seem to cut ok but if you do any staining on them they fall apart and any tissue left just doesn't look right - the nuclei look misshapen, fuzzy, stretched and extracted. In sections from the tissue stored in LN2 the nuclei were either missing or didn't take up the dapi at all (except at the very edge of the section). Has anyone had similar problems? I cut 10um sections, air dry overnight, fix 100% acetone 10min. Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Nuclear artefacts in FFPE tissues
I've been attempting to do some multi-colour fluorescence labelling of FFPE lymphoma tissue - usually I only use frozen tissue. It was all going smoothly on some FFPE tonsil sections but with the lymphoma tissues I see punctate nuclear staining with an epitope that should not be nuclear (CD32) - in some samples it appears as punctate staining all over the nuclei but in other samples it appears as a perinuclear halo. It only occurred with one particular antibody (rabbit anti-CD32) and not with rabbit anti-CD20 or mouse anti-CD68. Could this staining just be an artefact - is this common with FFPE tissue and is there something I can do to get rid of it. Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Background problems with frozen liver IHC
Hi All, I do a lot of fluorescence labelling of frozen mouse tissue (mostly spleens) but recently I've been trying to do IHC staining of various mouse (and human) tissues. I'm having particular trouble with liver samples with high brown background in the hepatocytes - the vascular areas are completely clean. I've been using Vector Labs avidin/biotin blocking system and their ABC detection method along with DAB (Dako) and various biotinylated secondary antibodies. There is background from the secondary antibodies and to a lesser extent from the ABC. Any tips? Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] PVP method for frozen bone sectioning
Hi all, After searching the internet I found a method for preparing mouse bones for frozen sectioning. I've been trying it out but have been having problems with the PVP coming out of solution (7.5% PVP-40T, 10% EDTA Na2 in 0.1M Tris.HCl pH6.95). I heated it to ~50-60oC to get it to dissolve but when its left in the fridge a ppt develops and the mouse femur that I have decalcifying in it is covered in large white blobs (however the decalcification does seem to be working ok). Does anyone have any experience of doing frozen bone sections this way? Any advice greatly appreciated. Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] ER labelling of fixed cells
Hi all, I'm trying to do some double labelling to show that a protein resides in the ER but I'm having some trouble getting nice reticulated ER staining in fixed cells. So far I have tried fixing in 4% PFA (15min on ice) followed by permeabilization with Tritonx100 (0.25%; 10min; room temp) or Saponin (0.5%; 7min; room temp; all susequent solutions containing 0.05% saponin). ER antibody is rabbit anti Calnexin (Stressgen). The labelling I'm getting is quite strong but is very punctate. Staining of my protein of interest is also very puntate but doesn't appear to colocalise with the clanexin and also I don't see any staining of the plasm membrane - which I should! Anyway the Prof wants to see nice reticulated staining of the ER - which I have seen in live cells with ER tracker but is it possible to get the same thing with fixed cells? I thought about trying to fix with glutaraldehyde but ?concentration ?time.. Also my PFA was in PBS would it be better to put it in HEPES or PIPES? Any suggestions welcome! Thanks Sonya ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
