We are searching a good anti-mouse pan Cytokeratin antibody (or CK18 mAb) for immunostaining of renal tubular epithelia in PFA-fixed paraffin sections. We tested different mAb (e.g. Clone AE1/AE3, AE1/AE3+8/18, etc.) but none of them worked on PFA-fixed paraffin kidney section from mice. We tested these mAb with cultured cells (from mouse and human) - and they worked. Does anyone know a good working mAb?
Dear Patrick,
Our lab is also having the same issue. I will be in touch if we happen to succeed. I gather the antigen retrieval may matter. See the link for the various broad cytokeratins, tissue specificity, IHC methods from the following link from Purdue IHC lab. You may need to scroll down to the cytokeratins from the list of antibodies. http://web.ics.purdue.edu/~jramosva/IHC%20tests/html/TemplateihctestsmainFrameset.htm
All the best and stay in touch if you happen to figure it out before we do. Best. Hibret
Hello,
I am searching a pan cytokeratin antibody for cryostat sections. I tested the clone AE1/AE3 antibody (Dako) and it worked pretty well on paraffin-embedded sections from mice but the other antibodies were opposite (for co-labelling). That's why I would like to ask if you know a cytokeratin works with cryostat sections. Thank you so much for your help.
Best. Huyen
Hi there, we haven`t solved our problem. Our mAb didn`t work on cryo- or paraffin-sections. Please tell me what protocoll you used on paraffin-sections - we also tested AE1/3 , but without a positve result.....
Hi,
It's here the protocol that I'm working for cytokeratin 19 (M3515, Dako) on mice sections.
Paraffin immunofluoresence protocol
· Deparaffinization :
o Xylène 1 10mn
o Xylène 2 10mn
o Ethanol Absolu 1 5mn
o Ethanol Absolu 2 5mn
o Ethanol 95 % 3mn
o Ethanol 70 % 1mn
o Ethanol 50 % 1mn
· Rince sections 1 time with tap water
· Rince sections 1 time with distilled water
· Pre-treatment : in water bath at 96°C, incubate slides in a solution of citrate buffer (Diapath) + 0.05% Tween20, or TRS6 (Dako), or TRS9 (Dako) for 20 min, then put them at room temperature for 30 min
Rince sections 3 times with PBS 3 min
Blocking with PBS/BSA 3% + 10% SFB + 10% Goat Serum ou Donkey Serum 1H at RT
Incubate with primary antibody in PBS/BSA 3% 1X ON at 4°C
· Rince sections 3 times with PBS 3 min
· Incubate with AlexaFluor 488 (green) or AlexaFluor 568 (red) conjuged secondary antibody 1/500 in PBS/BSA 3% 1H at RT (protected from light)
· Rince sections 3 times with PBS 3 min (protected from light)
· Rince sections 1 time with distilled water (protected from light)
· Mount sections using Prolong DAPI.
You should also notice about the secondary antibody. My colleague used the same primary Ab, the same protocol but it didn't work in her hand.
Good luck !
Thank you very much, we try this protocoll.....Can you please post the number of the secondary antibody (DAKO?, No. ??). Best regards Patrick
No, it is not from Dakol. It is anti-mouse secondary Antibody (IgG) from Life Technologies. Hope you will have good results.
Hi,
I have a quick question about the immunofluorescence protocol. Which reagent does the SFB stand for in the blocking step? Thank you!
Did anyone find a good Pan Cytokeratin antibody for IF protocols in mouse tissues? I am using formalin fixed tissues.
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