Additional file 4: Figure S3.

Optimization of the fixation and permeabilisation conditions for the anti-CXCR4 (ab2074) antibody in fMSC or HeLa cells. (A and B) fMSC or HeLa cells were fixed and permeabilised as follows (from left to right): methanol, 4% paraformaldehyde (PFA) + 0.3% Triton and PFA + 0.1% Tween, and then stained with CXCR4 (ab2074) as detailed in the methods section. The negative control was PFA + Triton treated cells but the primary CXCR4 antibody omitted. C) HeLa cells with the anti CXCR4 antibody clone ab2074 or 12G5, where the cells were fixed in PFA to show surface staining or treated with PFA + Triton to show intracellular staining. D and E) Fetal MSC and HeLa cells were transiently transfected with Rab5-GFP or Rab11-GFP constructs to ensure the accuracy of the Rab antibody staining. Note that Rab5-GFP shows classic punctate endosomal localization in both fMSC and HeLa, whereas the Rab11 shows diffuse cytoplasmic localization in fMSC and classical endosomal punctae in the HeLa cells, similar to antibody staining in Figure 2.

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Pelekanos et al. BMC Cell Biology 2014 15:15   doi:10.1186/1471-2121-15-15