I am trying to detect Pan-keratin by immunohistochemistry with a Sirius red counterstain. I have no problems with the Sirius red staining normally, however as soon as my sections have been exposed to antigen retrieval (any type of ag retrieval), the Sirius red no longer works and the whole sections turns bright pink.
Has anyone come across this problem before and managed to solve it? Any suggestions would be greatly appreciated.
Details:
Mouse formalin-fixed paraffin embedded tissue
Picrosirius red at 0.1%
Acetic acid wash at 0.5%
If your intention is to display cytokeratin versus collagen then I would suggest avoiding IHC and use Masson's Trichrome stain with methyl blue. Difficult to say more without knowing intention of study. Could also try Van Gieson counter stain (acid fuchsin rather than Sirius red).
Hello, I agree with Mr Galvan’s comment. Generally the couterstainings are performed as an end step of the immunostainings. Regards
Hi there, all !
I guess, there is some misunderstanding the question/ the background.
As I see the ,
Sirius red staining ("classical" HC- or counter-stain, = without / no antigen retrieval before IHC) usually ("staining normally") for Victoria is no problem.
But if the sections/material to be stained is subjected prior to Ag-Retr., sirius red will not stain selectively as usual but will stain all section substrate with pink color.
Right?
Thanks, Victoria, for clarifying this, best wishes and regards,
Wolfgang
Hi victoria
I agree with Damien that Masson's Trichrome method can give you satisfied results because ,in immunohistochemistry, counterstaining is always done for nuclei by Haematoxylin but not for the cytoplasm.
Hi all
If using IHC for cyto keratin I would likewise, as Gaber suggests, use Mayer's Hx or similar as counter stain but I am intrigued by Victoria's approach. Can you clarify if intention is purely to show keratin or if you are looking to show collagen as well?
Damien
Try to perform the microwave pretreatment after the Sirius Red and before the immunohistochemistry procedure.
Michael kasper
Hi, antigen retrieval could alter staining, as it is a chemical reaction as well as picro Sirius red staining is. I agree with colleagues suggesting to perform before the IHC with Ag retrieval, then picro Sirius red. Additional possibility is to label both markers with an antibody -based system
Hi Alanna,
I did get this technique to work. I would be happy to send you a copy of the protocol if you like? Basically, what I found is that heat induced antigen retrieval necessary for immune, damages the collagen fibres as the temperature is raised above 50C during the process. By shortening the antigen retrieval time (I heated on full power for 3 minutes) I was still able to get good quality immunostaining whilst being able to perform a standard Sirius red counter - 10-20 minutes in the picro-Sirius red solution, followed by 2 short acetic acid washes. Sirius red counterstaining also worked well with antibodies that require enzymatic digestion for the antigen retrieval - again based on the fact that the sections are not raised above 50C.
I hope this helps/ makes sense.
Regards,
Victoria
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology