Hi,
I'm trying to stain a viral infection of the pancreas in paraffin sections of adult (3 month old) BALB/c mice.
Prior to starting my histology analysis, I have titrated the organ to confirm that I actually have a viral load in the pancreas of my infected animals.
I have also used a organ (liver) were I know that I have abundancy of viral positive cells as a control for my work.
In my protocol I did Citrate Buffer retrieval (I have also tried Tris-EDTA) on 97C, used a commercial block of endogenous HRP, and 3%BSA to block nonspecific binding. My primary antibody is a nuclear marker, and is biotinylated (I have also tried un-biotinylated). My secondary antibody S-POD (also tried Envision for un-biotinylated Ab) and I use DAB as my chromogen.
When developing my chromogen I observed little no-specific staining, mostly in the areas that have dried. However, I did observed a lot of small brown DAB spots that mostly fade once I counterstain. And the biggest problem is that I didn't observe a single positive nuclear staining in 3 separated experiments. In each experiment I used pancreases from 3 different mice.
I observed a sufficient viral titer, which meant that the virus is present in the pancreas and specific positive cells with no background staining in my positive control which meant that the protocol and my work was ok?!
I know that the maybe I should think about changing my viral marker, however, this marker is mostly abundantly present in other tested organs.
I was wondering if the pancreas as a organ has some specific guidelines that I must follow, or if someone has some tips or tricks for pancreas staining in IHC!
Thank you for your help and suggestions,
Fran
Hi, Fran!
Did you try other IHC markers on pancreas parafin sections?
If other IHC methods do not work on your pancreas sections, the problem may be associated with the stage of sample preparation and fixation of the organ or further processing and embedding in paraffin.
If there is IHC staining for other markers, then the problem is in the antigen unmasking procedure (which sometimes happens when working with nuclear antigens). Try to introduce an enzymatic treatment step prior to thermal antigen unmasking. It would be helpful if you could indicate which antigen should be determined.
Best regards,
Artem
Hi, Fran!
Did you try other IHC markers on pancreas parafin sections?
If other IHC methods do not work on your pancreas sections, the problem may be associated with the stage of sample preparation and fixation of the organ or further processing and embedding in paraffin.
If there is IHC staining for other markers, then the problem is in the antigen unmasking procedure (which sometimes happens when working with nuclear antigens). Try to introduce an enzymatic treatment step prior to thermal antigen unmasking. It would be helpful if you could indicate which antigen should be determined.
Best regards,
Artem
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining