Anti-Cytokeratin 18 抗体 [C-04] (ab668)

Hello! Please find below our mouse on mouse staining tips. This may help you to improve your results. Additionally I would recommend you reduce the primary incubation to overnight, and incubate the antibodies in a solution of TBS plus 1% BSA.

We cannot vouch for the Millipore version of the clone but we have tested ab32118 and found that it does not react in mouse, and have placed this information on our datasheet to inform our customers.



Tips for eliminating background when staining mouse tissue with a mouse monoclonal



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Staining of mouse tissue using mouse antibody is a complicated process as high levels of background are often observed. It is notoriously difficult to eliminate this background.
Much of the background is caused by secondary antibody binding to endogenous mouse IgG in the tissue being stained, and to Fc receptors on B cells, plasma cells and macrophages.
Abcam is unable to guarantee monoclonal mouse antibodies on mouse tissue (unless stated on the datasheet). However, there are a few tips to try and reduce this background should mouse on mouse staining be necessary:

1. Blocking of endogenous IgG



Prepare tissue sections as usual.
At the usual blocking step, block with serum (from same species as the secondary antibody) for 30 min at room temperature.
Wash 3 X 2 min with PBS Tween 20.
Incubate sections with an unconjugated AffiniPure Fab fragment Anti-Mouse IgG (H+L) for 1 hr at room temperature, or overnight 4°C . Try this blocking antibody at 0.1 mg/ml although the optimal concentration will need to be assessed by the end user.Proceed with antibody staining.




2. Blocking endogenous Fc receptors
There are kits available for this which use F(ab) monomeric secondary antibodies.

Other tips that can be used to decrease general background:


Incubate sections with 1% Triton (in PBS) at room temperature for 30 min to ‘clean’ the tissue.
Use TBS –Tween 20 as a washing buffer. This often gives less background than using PBS-Tween 20.

Thank you for your reply.


I am sorry to hear that you have difficulties getting satisfactory results form this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab668.

1.) I suggest to decrease the amount of Triton X used in the buffers. 0.025% Triton X-100 is used in standard protocols. Triton X is a quite harsh detergent.

2.) Secondary anti pan IgG antibodies sometimes bind some isotypes better than others. I therefore suggest to try a different secondary antibody and use an IgG1 specific antibody.

Please let me know how you are getting on.