[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] 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 frag

[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

[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

[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) -

[Histonet] Cytoplasmic staining with Ki67 in frozen sections

HI All, I've just been trying out a Ki67 antibody from Abcam (rabbit polyclonal) on some frozen mouse tissues. Staining looked ok in the colon with nuclear localisation in the cells of the basement membrane. However, what I'm really interested in is the spleen and the staining here was a mess!

[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 bu

[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 com

[Histonet] Spleen EM images

Hi there, Just wondering if there are any EM experts out there who could look at some EM of mouse spleen sections for me and annotate them. I do a lot of LM work on spleen sections and I had the chance to try some EM but unfortunately nobody could help me identify what sort of cells I was looki

[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