I am optimizing an immunocytochemical protocol for the detection of the circulating tumor cells (CTCs) from metastatic breast cancer with CKAE1/AE3. The cells were previously fixated in Delaney (PAP) or methanol. Unfortunately, with everything that I've tried (changing the dilution, fixation, detection kits, adding an amplification...), the CTCs remain unstained (my positive control is always positive).
Do you have any suggestions on what else can be done for my CTCs to bind the CK antibody?
Dear Simona!
Please You look at following article and protocol:
Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer
https://www.nature.com/articles/bjc2015132
Cultivation of cell lines. MDA-MB-231 cells
Immunocytochemical double staining was performed using anti-pan cytokeratin (panCK) antibodies (AE1/AE3, Dako) together with two different anti-PLS3 antibodies (C15, Santa Cruz Biotechnology (Plastin antibody 1) or PA5-27883, Thermo Scientific (Plastin antibody 2)). Briefly, slides were thawed 30 min before incubation with 4% PFA in PBS. After washing with PBS, cells were permeabilised using 1% Triton X-100 in PBS. Blocking of unspecific binding was performed after two washing steps using 10% AB-Serum (Biotest, Dreieich, Germany) in PBS. Anti-PLS3 antibodies were then added at a dilution of 1 : 100 (10% AB-Serum in PBS) and incubated for 45 min. Subsequently, slides were washed with PBS, and Alexa546 goat anti-rabbit secondary antibodies (Molecular Probes, Eugene, OR, USA) were applied for 45 min (diluted 1 : 200 in 10% ABSerum in PBS). After further washing steps, the panCK cocktail (AE1/AE3) was applied (1 : 300) and incubated for 45 min, followed by additional washing steps. Slides were covered with Vectashield Mounting Medium containing DAPI (Vector Laboratories, Burlingame, CA, USA).
Hi,
the protocol from Alexandr is a good starting point. The absent staining in your samples may originate from the missing permeabilization (at least you did not state about this). One question would be: How did you define the presence of CTCs? How many cells did you analyze?
Best,
Norman
How do you know if the cells you are trying to visualize are epithelial (CTCs) and not histiocytes or other hematopoietic cells? Perhaps share your images with a counterstain
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus