HI Alexis,

I suspect the processing and embedding of your tissues will seriously affect your ability to detect CD11c.

Routinely we perform CD11c (Itgax) labelling on cryosections from flash-frozen tissues. Even then you usually only detect CD11cHi cells. Using a signal amplification method (TSA kit or similar) and avoiding the commonly used serum blocking (which we found seriously reduces antibody binding to CD11c) you can commonly detect cells that are CD11cLo (by flow cytometry).

For examples of our staining see our publication: Defining the anatomical localisation of subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax, CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages http://www.sciencedirect.com/science/article/pii/S0171298511001860

Unfortunately we did not publish CD11c staining in lung tissue, but we show CSF1R+ cells (CSF1R:eGFP) with and without CD11c based deletion using CD11c:DTR mouse models showing there is clearly a CD11c negative subset. Details on splenic subsets can be found in the paper. If you need more detailed protocols please feel free to get in touch.

Kind regards, Barry

The picture on the left side shows a stained cell that looks like a plasm cell. Or your antibody or your technique is not working.

I suggest you to make the standardization using lymph node (a tissue rich in CD11c+ cells). Shift to the lungs and spleen only after to have sure that you are able to identify CD11c+ cells in the lymph node.

If your objective is only to identify CD11c+ cells, maybe you should use immunohistochemistry. This is simpler than IF and you can analyze the morphology of CD11c+ cells to exclude plasm cell positivity (false-positive).

Good luck!

With respect to the red labeling it seems to be rbc's. Could you add some additional information regarding the primary and secondary antibodies used: which species are they raised in and do you have some positive indications that the primary antibody works in mice?

Hi,

It appears you are getting some signals of Macs in the lung region! You might want to distinguish them from other lung cells. I would suggest you to use T1 alpha (pneumocyte-type 1 cells), SPC (pneumocyte-type 2 cells) or FSP1 (fibroblast) antibodies. You have to standardize the working dilution of primary and secondary antibody for your sections as fixation and processing generally differs. The punctate signals you are observing is quiet real, since you are scanning by confocal. Try increasing the pinhole, your signal will become little brighter. Depending on what treatment the mice have gone through, it is always good idea to have control mice. Keep the same setting to scan the controls and the experimental mice to distinguish the difference.

Hope it helps!

The red is just the background and not a label at all. the only "true" signals are those FITC dots which look like "clusters" but seem not exclusive cell surface. For ab choice, you may want to look into if the clone is tested for FC, IHC, or IHC-F, frozen or paraffin etc, and you should choose the one good for IHC-F that works for paraffin the best. Company like BD would provide those info. For positive control, simplest one you can use WEHI274-1(?) a mouse monocytic cell line for cytospin and formalin fixed. You should be able to see a nice "green ring" around the cell contour or a partial ring with clusters like "green diamond" around if activated before fixed. Good luck with your pursuit.

HI Alexis,

I suspect the processing and embedding of your tissues will seriously affect your ability to detect CD11c.

Routinely we perform CD11c (Itgax) labelling on cryosections from flash-frozen tissues. Even then you usually only detect CD11cHi cells. Using a signal amplification method (TSA kit or similar) and avoiding the commonly used serum blocking (which we found seriously reduces antibody binding to CD11c) you can commonly detect cells that are CD11cLo (by flow cytometry).

For examples of our staining see our publication: Defining the anatomical localisation of subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax, CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages http://www.sciencedirect.com/science/article/pii/S0171298511001860

Unfortunately we did not publish CD11c staining in lung tissue, but we show CSF1R+ cells (CSF1R:eGFP) with and without CD11c based deletion using CD11c:DTR mouse models showing there is clearly a CD11c negative subset. Details on splenic subsets can be found in the paper. If you need more detailed protocols please feel free to get in touch.

Kind regards, Barry

Mouse CD11c staining usually works fine on both frozen and paraffin sections. We did different tissues and used cytospin cells as a control. Staining is on the surface of DC and their long "dendrits" can be seen aroung, since they are often cutted. Some activated Mf might be also positive. There are a lot of suppressor DC in the lung and they are often CD11c low.

Have you tried antigen retrieval technique?

Your signal looks non-specific. Lung alveolar macrophages also have a lot of autofluorescence. Regarding CD11c staining, we have looked hard and could not find an antibody that works in paraffin. But OCT sections work great, see Koltsova,E.K., Garcia,Z., Chodaczek,G., Landau,M., McArdle,S., Scott,S.R., von Vietinghoff,S., Galkina,E.V., Miller,Y., Acton,S.T. et al 2012. Dynamic T cell-APC interactions sustain chronic inflammation in atherosclerosis. J. Clin. Invest. 122:3114-3126. Good luck!

I think that the cell containing green granular structure at the left side looks like alveolar macrophage or mast cell. On the other hand, it is clear that red stained cells are erythrocytes and blue stained cells are leukocytes in order to their shape. However, your staining is very bad because all the signals are too weak except green signals. ıf you try again of your stain you should increase your primary antibody incubation time and reduced your time under light in case of excess flourescent light absorbtion. At the same time you should notice that IF in paraffin sections is too diffucult than IF in cell culture or IHC in paraffin sections. So you should care that antigen retrieval must be made sufficiently. I believe that some repeats will solve your problem

What secondary did you use? For how long? Have you experimented with different concentrations and times?

@michael Shurin:

What clone/pAb did you use, can you share your protocol?

our experience has been similar to Klaus Ley's

The general consensus is that CD11c isn't retained following paraffin embedding. I know that CD11c can stain PFA fixed immune cells very well, but I am not sure about frozen tissues. The smaller red signals come from the autofluorescence of blood cells in surrounding blood capillaries. Two possibilities are there: either your signal gain is so high that you are seeing background autofluorescence in your alveolar macrophages instead of a true CD11c signal (in which case, there is no difference between your CD11c+ FITC and hamster IgG + FITC negative control), or that you do have CD11c staining - but surface CD11c signal is too low, and only endosomes are being stained.

We are trying out CD11c on frozen sections, so I will keep you informed if we see anything.

Hello Mubing, I am interested in what you mean by saying "CD11c isn't retained following paraffin embedding" but at the same time "CD11c can stain PFA fixed immune cells very well". PFA fixation is paraformaldehyde --> paraffin, no?

My group also wants to stain (human thymus) tissues with anti-CD11c antibodies and visualise using streptavidin-HRP-DAB.

The most traditional PFA fixed and non-paraffin embedded method would be be cryosectioning (but frozen sections are very difficult to cut, and there is a loss of architecture - the upside is that better antigen preservation exists so sometimes people are forced to go down this route). We are also actually exploring other ways in the lab of staining PFA-fixed lung tissue (cutting 150 um thick agarose gel embedded sections on a vibratome for example).  

It sounds like your samples are all paraffin embedded samples though. Unfortunately, I have come across very few papers with CD11c staining in paraffin sections (I have seen them for MHC Class II though in mice, could this be an alternative for you?). I have seen CD11c staining for cryosections though (http://emboj.embopress.org/content/31/10/2378), which makes me suspect it is an antigen which gets masked or destroyed by the embedding process.

I've attached some old staining of mine on CD11c staining of PFA fixed alveolar macrophages ex vivo undergoing phagocytosis. The blue is a typical pattern of CD11c staining seen (on the surface rather than internally - in this experiment I tried to fit 5 markers into a 4 colour confocal microscope, so my DAPI/BV421 channel had both Hoerchst internal and CD11c external staining).